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" heat shock transcription factor1 hsf1 was overexpressed to promote glutaminolysis and activatemtor in colorectal cancer crc here we investigated the mechanism for cancerspecific overexpression of hsf1methods hsf1 expression was analyzed by chromatin immunoprecipitation qrtpcr immunohistochemistrystaining and immunoblotting hsf1 translation was explored by polysome profiling and nascent protein analysisbiotin pulldown and m6a rna immunoprecipitation were applied to investigate rnarna interaction and m6amodification the relevance of hsf1 to crc was analyzed in apcmin and apcmin hsf1miceresults hsf1 expression and activity were reduced after the inhibition of wntcatenin signaling by pyrvinium orcatenin knockdown but elevated upon its activation by lithium chloride licl or catenin overexpression thereare much less upregulated genes in hsf1ko mef treated with licl when compared with licltreated wt mefhsf1 protein expression was positively correlated with catenin expression in cell lines and primary tissues aftercatenin depletion hsf1 mrna translation was impaired accompanied by the reduction of its m6a modificationand the upregulation of mir4553p which can interact with ²utr of hsf1 mrna to repress its translationinterestingly inhibition of mir4553p rescued catenin depletioninduced reduction of hsf1 m6a modificationand mettl3 interaction both the size and number of tumors were significantly reduced in apcmin mice whenhsf1 was genetically knockedout or chemically inhibiteds catenin suppresses mir4553p generation to stimulate m6a modification and subsequenttranslation of hsf1 mrna hsf1 is important for catenin to promote crc development targeting hsf1 could bea potential strategy for the intervention of catenindriven cancerskeywords colorectal cancer catenin hsf1 translation mir4553p m6a rna modification correspondence wangx118zjueducn jinhczjueducn1department of medical oncology cancer institute of zhejiang university sirrun run shaw hospital school of medicine zhejiang university hangzhouchina2labortary of cancer biology key lab of biotherapy in zhejiang sir run runshaw hospital school of medicine zhejiang university hangzhou chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csong molecular cancer page of introductioncolorectal cancer crc is the third most common cancer with high mortality rate globally the accumulation of various genetic and epigenetic changes activatesmultiple oncogenic signaling critical for the pathogenesisof crc such as wntcatenin signaling pathway its activation will eventually initiate a transcriptiondependent oncogenic process to promote cell cycle progression and apoptosis resistance while the mechanismfor activated wntcatenin signaling to promote crcdevelopment has been wellexplored no therapeuticstargeting this pathway has been successfully developedin addition to proliferation activation and apoptosisresistance metabolism reprogramming is one of important hallmarks of cancer cells for example cancercells favor glycolysis instead of oxidative phosphorylationfor glucose metabolism even in aerobic conditionswhich was wellknown as warburg effect [ ] pyruvate the last product of glycolysis is converted into lactate rather than acetylcoa acetyl coenzyme a fortca tricarboxylic acid cycle or the krebs cycletherefore targeting enhanced glycolysis has been proposed as novel options in the prevention and treatmentof human cancers including crc however as a metabolism hub tca cycle is important in both energyproduction and biosynthesis therefore it needs to bereplenished by anaplerotic reactions such as glutaminolysis previously we reported that heat shock transcription factor hsf1 stimulated glutaminolysis toactivate mtor and promote crc development by upregulating the expression of glutaminase gls1 thecritical enzyme in glutaminolysis hsf1 expressionwas increased in crc and had a positive correlationwith shorter diseasefree survival dfs however theupstream mechanism for hsf1 overexpression in crcwas still uncleargene expression can be controlled by multiple processesincluding transcription mrna degradation translationand protein degradation while gene translation and protein degradation have been extensively investigated moreand more studies focused on mrna translation by exploring the effect of noncoding rnas such as micrornasmirnas and new modifications of mrna including n6methyladenine m6a modification [ ] mirnas canform a mirnainduced silencing complex mirisc toposttranscriptionally regulate gene expression by inhibiting capdependent initiation and stimulating mrna deadenylation [ ] on the other hand as one of the mostabundant modifications in mrna m6a modification ofmrnas usually promotes translation by recruiting initiation factors such as eif3 to the ² end of the mrna while mirnas and mrna m6a modifications play a distinct role in mrna translation the interplays betweenthem were not clarifiedin this study we found that activated wntcateninsignaling stimulated hsf1 translation to promote crcdevelopment byrepressing hsf1 mrnatargetingmir4553p to increase m6a modification of hsf1mrna therefore targeting hsf1 translation could be anew strategy for the intervention of crc and other cancers driven by activated wntcatenin signalingmaterials and methodscell antibodies and chemicalshuman crc celllines sw480 sw620 dld1 rkowere obtained from the american type culture collection atcc all cells were routinely cultured in rpmi invitrogen or dmem invitrogen supplemented with fetal bovine serumall cells were incubated at °c with co2 and humidity the following antibodies were used for western blotting and ihc hsf1 12972s cell signalingtechnology cst ab52757 abcam catenin 8480scst actin l cst flag f1804 sigmacyclind1 ab134175 abcam cleaved parp1 9541scst mettl3 a8370 abclonal gls1 ap8809babgent pyrvinium p0027 licl cycloheximide r750107 chloroquine c6628 mg132 and pd150606 d5946 were purchased from sigmaaldrichinterfering rna sirnasirna mirna mimicsinhibitors transfectiontargeting cateninsmallmettl3 and micrornas were synthesized by genepharma shanghai china and ribobio guangzhouchina the sequence of these sirnas and mirnaswere listed in additional file table s1 sirnas andmirna mimicsinhibitors were transfected into cellsseeded overnight by lipo2000 invitrogen usa or lipofectamine rnaimax transfection reagentinvitrogenusa according to the manufacturers instructionsluciferase activity assaythe plasmid of catenin reporter was gifted from profximei wu zhejiang university for hsf1 activity assaya fragment containing x hse were synthesized andinserted into the pgl3basic vectors promega corporation usa the plasmid was cotransfected with prlrenilla and catenin sirna by using lipo2000 invitrogen usa or treatment with pyrvinium by xtremegene hp dna transfection reagent roche usa ²utr segment of the hsf1 was cloned by pcr andinserted into the vector pmirreporter promegathe mutation of mir455 binding sites in hsf1 ²utrwas generated by quick sitedirected mutagenesis stratagene usa the resultant plasmidswere cotransfected with prl renilla and mir455 mimicsby usinglipo2000 invitrogen usa h post 0csong molecular cancer page of transfection the luciferase activity was measured by thedualglo luciferase assay system promega corporation usachromatin immunoprecipitation chipchip analysis was conducted with the simplechip¢ enzymatic chromatin ip kit cst usa antibodies usedwere antihsf1 12972s cst tcf7l2c48h11 cstand flag f1804 sigma the primers used for thepcr analysis of precipitated dna were shown inadditional file table s2 for flagchip assay theflagcatenin vector was transiently overexpressed bytransfection after h the enrichment of flagcateninon col27a1 promoter was measured by the chip kitimmunoblotting and immunohistochemistryimmunoblotting and immunohistochemistry ihc assays were performed as previously reported for immunoblotting total proteins were extracted with ripabuffer supplemented with protease inhibitors rocheusa after heating the protein sample to °c for min celllysates were transferred to polyvinylidenefluoride pvdf membranes after the membranes wereblocked in milk primary antibody with gentle agitation overnight at °cfor ihc assay was performed in a tissue array containing cases of colonic tissues primary antibodies usedwere listed above the degree of immunostaining wasassessed by independent pathologists and evaluated byassigning a score of scores were defined as follows no staining faint staining moderate staining and strong staining final scores of and were regardedas low expression whereas scores of and consideredas high expressionapoptosis detectioncell apoptosis was measured by flow cytometry analysisand western blotting for flow cytometry analysis ofapoptosis cells were harvested and resuspended in μl x binding buffer μl fluorescein isothiocyanatefitc annexin v and propidium iodide pi bd biosciences usa were added to the cell suspensionand then incubated for min at room temperatureafter that the samples were attenuated with 400ul xbinding buffer and analyzed by acs caliburflowcytometerpuromycinlabellingto detect the change of nascent hsf1 synthesis x cells were plated in cm dish afterthe giventreatment cells were incubated with biotindcpuromycin nu925bios jena bioscience for hcells were lysed with np40 buffer mm trishclph mm nacl np40 glycerolcontaining 1x protease inhibitor cocktail after adequatecentrifugation the supernatant was incubated with 80ulstreptavidin sepharose beads ge17 sigma byrotating at °c for h to overnight the mixture waswashed by np40 buffer for times and subjected towestern blotting using hsf1 antibodyseparatestranslatingpolysome profilingpolysome profilingor nontranslating mrnas on a sucrose gradient according tothe number of bound ribosomes as previously described in brief cells were grown to confluence before collection cells were incubated with μgml ofcycloheximide for min then cells are lysed by polysome buffer [ mmoll kcl mmoll mgcl2 triton x100 μgml cycloheximide mmoll heparin and uml rnase inhibitor takara 1x cocktail] for min on ice lysates were centrifuged rpm for min and the supernatant was layered onto a to sucrose gradient gradients were then centrifuged at rpm for min at °c and polysomebound fractions were collected using an isco densitygradient fractionation system isco lincoln ne withcontinuous monitoring based on a260nm wavelengththe rna in each fraction was extracted using trizol reagent invitrogen and analyzed by realtime rtpcrbiotin pull down assaybiotin pull down assay was performed as described previously cells were transfected with biotinylatedmir4553p probes for h and resuspended using lysisbuffer mm tris ph mm nacl mmmgcl2 uml superasein mm dtt igepal protease inhibitors lysates were incubated withprepared streptavidin beadsge healthcare yeasttrna sigma was used for blocking lysates at °c for h then washed times with binding and wash buffer mm trishcl ph mm edta m naclfinally the bound rnas were extracted and purified forqpcrrna immunoprecipitation rip assayrip assay was performed by magna riptm rnabinding protein immunoprecipitation kitmilliporeno17 briefly à cells were lysed in μlrip lysis buffer and immunoprecipitated with antibodiesofinterest and protein g magnetic beads at °covernight followed by six times of washes in washingbuffer and protein digestion at °c total rna wasisolated and subjected to rtpcr analysis followingantibodies were used for rip n6methyladenosine synaptic mettl3 a8370 abclonal igg millipore 0csong molecular cancer page of rnasequencing2x106 wt mef and hsf1 ko mef were plated andcultured overnight following day cells were treatedwith mm licl for h cells were collected with trizol reagent the total rna was processed by nebnext®polya mrna magnetic isolation module to enrichmrna and the product rna was used for constructionlibrary via kapa stranded rnaseq library prep kitillumina sequencing libraries denatured by mnaoh to generate singlestranded dna as amplified insitu illumina cbot truseq sr cluster kit v3cboths gd4013001 illumina the ends of the generatedfragments were used to run cycles by the illuminahiseq sequencer all the experimental steps afterthe rna extraction were conducted in kangcheng biotechnology co ltd aksomics shanghai china rnasequencing was performed three timesanimal experimentsanimal care and experiments were conducted in compliance with institutional animal care and use committeeand nih guidelines the c57bl6 j mice and apcminmice were purchased from model animal research center of nanjing university marc nanjing chinahsf1 ko mice reported previously were used to generate apcmin mice hsf1 subsequently groupsof mice wild type apcmin apcmin hsf1 and apcmin treated with knk437 as previously reported were fed with highfat diet kcal fat for monthsthe intestine was dissected flushed with pbs and cutopen longitudinally along the main axis the number oftumors was counted and the sizes oftumors weremeasuredstatisticsall data were expressed as mean ± sd unless specifiedthe students ttest was performed for statistical significance analysis p value was considered as statistically significantresultscatenin activates hsf1 in crcin an effort to explore potential regulations of hsf1 wescreened chemicals generating a gene expression patternsimilar to hsf1 depletion by connective map httpportalsbroadinstitutecmapinterestingly a recently reported inhibitor of wntcateninsignaling pyrvinium had a similar effect on genomewide gene expression as hsf1 depletion fig 1aand additional file figs1ab moreover the expression signature related to wntcatenin signaling was positively correlated with the hsf1 signature fig 1b indicating a potential connection of hsf1 withwntcatenin signaling indeed pyrvinium attenuated[ ]the activity of a luciferase reporter driven by hsf1 binding sites hse heat shock response elements [ ]fig 1c and reduced the expression of wellknown transcriptional targets of hsf1 such as hsp90aa1 hspa4hspb1 and hsph1 fig 1d and additionalfile figs1c chromatin immunoprecipitation chip assayfurther confirmed the reduced interaction of hsf1 withits transcriptional targets fig 1e and additional file figs1d in consistence with pyrvinium knockdown ofcatenin by sirna also decreased hsf1 activity fig1f reduced the expression of hsf1 targets fig 1g andadditional file figs1e and attenuated the interactionof hsf1 with its targets fig 1i and additional file figs1f furthermore hsf1 targets were upregulated bythe potent gsk3 inhibitor licl in colorectal cancer cellline rko which had a low level of catenin expressionfig 1hto explore the biological relevance of hsf1 activationto catenin signaling we profiled gene expression ofwild type mouse embryonic fibroblasts wt mef andhsf1 knockout mef hsf1 ko mef before and afterlicl treatment ncbi geo gse151119 while only genes were upregulated in licl treatedhsf1 komef there were genes significantly upregulated inwt mef after licl treatment fig 1j among them genes displayed a dependence on hsf1 since theirexpression levels failed to be upregulated by licl treatment once hsf1 was depleted fig 1k in fact their expression levels had a high correlation with theexpression of a previously reported hsf1 signature fig1l furthermore genes had a hse heatshock response element hse within their promoter regions fig 1m and additional file meaning that theyare most likely bona fide targets of hsf1 indeed someof them such as tma16 dedd2 hspa9 and kif21a havebeen confirmed as the target of hsf1 by chipseqncbi geo gse57398 fig 1n and additional file figs1g taken together these results indicated that catenin can positively regulate hsf1catenin stimulates hsf1 protein translationto delineate how catenin regulates hsf1 we quantitated protein levels of hsf1 before and after inhibitingcatenin both pyrvinium and catenin depletion reduced the protein level of hsf1 fig 2a and b in contrast overexpression of exogenous catenin increasedhsf1 protein level fig 2c furthermore hsf1 proteinlevel was increased after activating wntcatenin signaling by licl treatment in both rko and mef cellsfig 2d in addition hsf1 expression correlates wellwith catenin expression in primary tissues fig 2e andf p chisquare test all of these data indicatedthat catenin upregulates hsf1 protein expression 0csong molecular cancer page of fig wntcatenin signaling activates hsf1 a chemicals influencing gene expression in a similar manner to hsf1 inhibition were screened byconnective map analysis b the correlation of wntcatenin signaling signature and hsf1 signature was detected by gepia c the effect ofpyrvinium on hsedriven promoter activity was explored by luciferase reporter assay d the effects of pyrvinium on the targets of hsf1 wereanalyzed by rtpcr e binding of hsf1 to the promoters of hsf1 targets in crc cells treated with or without pyrvinium was determined by chipf the luciferase assays of hse before and after catenin knockdown were shown as in c g and h the mrna levels of hsf1 targets with catenin knockdown or licl treatment were analyzed by rtpcr i binding of hsf1 to its targets promoter in crc cells before and after cateninknockdown was analyzed by chip j volcano plot displays differentially regulated genes in dhsf1 compared to wt parental cells with licl reddots indicate significantly regulated genes based on adjusted pvalue and logfold change logfc p log2fc k differential geneexpression analysis in wt and hsf1 mef treated with licl were performed by rnaseq numbers of upregulated genes in two cells wereshown in venn graph l the correlation of putative hsf1dependent genes from k with reported hsf1 signature was detected by gepia mnumbers of putative hsf1dependent genes with or without hse in their promoters were summarized n representative hsf1 chipseqtracks ncbi geo gse57398 for hsecontaining genes are shown asterisks indicate p 0csong molecular cancer page of fig catenin stimulates hsf1 protein translation a the effect of pyrvinium on the protein expression of hsf1 was explored by westernblotting b the effect of catenin knockdown on hsf1 protein level was analyzed by western blotting c the protein level of hsf1 before andafter catenin overexpression was analyzed by western blotting d the effect of licl on hsf1 protein level in rko and mef was analyzed bywestern blotting e the expression of catenin and hsf1 in colorectal tissue was analyzed by immunohistochemistry staining f the correlationbetween catenin expression and hsf1 expression in colorectal tissue was analyzed by chisquare test p g the effect of catenindepletion on hsf1 with puromycin labeling was determined by western blotting h amount of hsf1 mrna in various polysome fractions wasanalyzed by rtpcrp however there were no apparent alterations in hsf1mrna level after catenin knockdown or pyrviniumtreatment additional file figs2a and s2b meanwhile the halflife of hsf1 protein was also not changedbefore and after catenin knockdown additional file figs2c inhibitors of proteasome autophagy and calpains all failed to reverse hsf1 protein downregulationinduced by catenin knockdown additionalfile 0csong molecular cancer page of figs2d all of these results implied that catenin affects hsf1 protein expression mostlikely via translationregulation therefore puromycin labeling assay wasemployed to monitor the synthesis of nascent hsf1 protein as expected the puromycin labeling of hsf1was reduced by catenin depletion fig 2g to furtherconfirm it monopolysome fractions from cytoplasmicextracts of crc cells before and after catenin depletion were collected by sucrose gradient centrifugationthe subsequent rtpcr analysis revealed that catenindepletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 2h in summary catenin upregulates hsf1 expression by stimulatinghsf1 protein translationhsf1 protein translation is regulated by mir4553pas micrornas mirnas play an important role inregulating the efficiency of protein translation we wondered whether hsf1 protein translation was regulatedby micrornas based on bioinformatics screening bytargetscan mirdb and starbase some micrornas including mir4553p mir2145p mir4315p mir184mir4903p and mir375 were proposed to target ²utr of hsf1 mrna fig 3a after functional validation by western blotting mir4553p and mir2145pbut not other micrornas were capable to suppress theexpression of hsf1 protein in crc cells fig 3b andadditional file figs3a however mir2145p but notmir4553p also reduced hsf1 mrna level additionalfile figs3b whats more an inhibitor of mir4553prather than mir2145p rescued the downregulation ofhsf1 protein by catenin knockdown fig 3c and additional file figs3c indicating that mir4553p mightbe relevant to catenininvolved regulation of hsf1protein translation indeed mir4553p inhibited the activity of luciferase driven by wild type hsf1 mrna ²utr but not its mutant unable to bind mir4553p fig3d the interaction of mir4553p with hsf1 mrnawas further confirmed by biotin pull down assay fig3e based on the analysis of tcga data httpmirtvibmssinicaedutwthe expression of mir4553p islower in colon adenocarcinoma than in normal tissuesadditional file figs3d similarly qpcr analysis revealed lower levels of mir4553p in crc tissues than inadjacent nontumor tissues fig 3f additionally wehad confirmed high expression of hsf1 in the same cohort of human crc tissues previously indeedmir4553p expression was negatively correlated with theexpression of hsf1 fig 3g on the other handmir4553p similar to hsf1 inhibition as we reportedrecently reduced the expression of hsf1 targets induced the viability inhibition and apoptosis activation ofcolorectal cancer cells fig 3hj and additional file figs3eg the seed sequence of micrornas was important for targeting mrna by basepairing indeed the seed sequence mutant of mir4553p could notdownregulate the protein level of hsf1 additional file figs4a confirming the importance of mir4553p totarget hsf1 protein expression in a word mir4553ptargets hsf1 mrna ²utr to inhibit its translationm6a modification of hsf1 mrna stimulates its proteintranslationin addition to microrna mrna modifications such asn6methyladenosine m6a play important roles in theregulation of hsf1 translation interestingly we noticedthat the matching sites of mir4553p seed sequence inhsf1 mrna ²utr contains a typical motif of m6amodification fig 4a which was supported by bioinformatic analysis httpwwwcuilabcnsramp fig 4b andadditional file figs4b moreover we have done themerip sequencing in sw620 and found that the ²utr region of hsf1 has one m6a modification site intriguingly this sequence is completely complementary tothe seed sequence of mir4553p additionalfile figs4c pcr analysis after merip m6a rna immunoprecipitation further confirmed m6a modification ofhsf1 mrna fig 4c whats more the activity of luciferase driven by the mutant hsf1 mrna ²utr whichwas unable to bind mir4553p but retains the m6amodification site sequence drach d a g or u r a or g h a u or c [] was higher than theactivity of luciferase driven by wild type hsf1 mrna²utr fig 3dindicating the importance of m6amodification to hsf1 expression as the main component of the methyltransferase writer complex [ ]mettl3 was also bound to hsf1 mrna fig 4donce its expression was depleted m6a modification ofhsf1 mrna was decreased fig 4e in consistencewith its potential roles in promoting protein translationsuch a reduction of hsf1 mrna m6a modification reduced hsf1 protein expression fig 4f and nascenthsf1 protein synthesis fig 4g furthermore mettl3depletion considerably reduced the presence of hsf1mrna in the polysome fraction but increased in nontranslating ribosome fractions fig 4h while hsf1mrna or the stability of hsf1 protein were not changed additionalfile figs4d and s4e moreoverhsf1 protein was decreased with the knockdown ofythdf1 which was the reader protein of hsf1 m6amodification fig 4i to sum up m6a modification ofhsf1 mrna was relevant to stimulate its translationcatenin suppresses mir4553p to increase hsf1 mrnam6a modificationnext we explored the interplay between mir4553p andm6a modification of hsf1 mrna both hsf1 mrna 0csong molecular cancer page of fig hsf1 protein translation is regulated by mir4553p a overlap of hsf1targeting micrornas predicted by targetscan mirdb and starbaseb the effect of mir4553p on hsf1 protein was analyzed by western blotting c the effect of mir4553p inhibitor on catenin knockdowninduced hsf1 downregulation was determined by western blotting d luciferase activity assay was used to analyze the effect of mir4553p onthe activity of ²utr with or without mir4553p binding sites p e the binding between biotinmir4553p and hsf1 mrna wasdetermined by biotin pull down assay p f expression of mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed by qpcr g the correlation of hsf1 protein and mir4553p in pairs of fresh crc tissues and adjacent nontumor tissues wasanalyzed h the effect of mir4553p on viability of crc cells was explored by mts assay i the effect of mir4553p on apoptosis of crc cells wasanalyzed using flow cytometry after pi and annexin vfitc double staining j apoptosis of crc cells treated with or without mir4553p wasdetermined by western blottingm6a modification and its binding to mettl3 were decreased by the overexpression of wild type mir4553pbut not its mutant unable to bind to hsf1 mrna ²utr figfigs5aand additional5afileinterestingly mettl3 depletion not only reduced m6amodification of hsf1 mrna fig 4e but also enhanced the interaction of mir4553p with hsf1 mrnafig 5b and additionalfile figs5b while the 0csong molecular cancer page of fig m6a modification of hsf1 mrna stimulates its protein translation a the sites of hsf1 ²utr binding to the seed sequence of mir4553pwas consistent with m6a rna modification elements drach b bioinformatic prediction of m6a modification in ²utr of hsf1 mrna c m6amodification of hsf1 mrna was analyzed by merip p d binding of mettl3 to hsf1 mrna was detected by rip p e m6amodification of hsf1 mrna with or without mettl3 depletion was analyzed by merip p f the protein level of hsf1 before and aftermettl3 depletion was detected by western blotting g the effect of mettl3 knockdown on hsf1 synthesis was determined by western blottingafter puromycin labeling h amount of hsf1 mrna in various polysome fractions was analyzed by rtpcrp i the effect of ythdf1knockdown on hsf1 protein level was analyzed by western blottingexpression of mature and primary mir4553p was notupregulated additional file figs5cd these resultsindicated that mir4553p may compete with mettl3for the m6a modification of hsf1 mrna thus inhibitinghsf1 protein translation furthermore the binding ofmir4553p to hsf1 mrna was not changed by ythdf1deletion additional file figs5e indicating that thetranslation repression of hsf1 mrna was more likely tobe mediated directly by the reduced m6a modification ofhsf1 0csong molecular cancer page of fig catenin suppresses mir4553p to increase hsf1 mrna m6a modification a m6a modification and mettl3 interaction of hsf1 mrnawith wt or mutant of mir4553p were analyzed by rip b the interaction between biotinmir4553p and hsf1 mrna with or without mettl3depletion was analyzed by biotin pull down c the effect of mir4553p inhibitor andor catenin knockdown on m6a modification of hsf1mrna was analyzed by merip d the effect of mir4553p inhibitor andor catenin knockdown on the interaction of mettl3 with hsf1 mrnawas analyzed by rip e the effect of catenin knockdown on interaction of mir4553p and hsf1 mrna was analyzed by biotin pull down f andg the levels of mature f and primary g mir4553p with catenin knockdown or licl treatment were determined by rtpcr h the correlationof col27a1 and mir455 was analyzed in linkedomics httplinkedomics i the effect of catenin depletion or licl on mrna level ofcol27a1 was analyzed by rtpcr j the interaction of catenintcf7l2 and hsf1 promoter was determined by chip k the correlation of catenin protein expression with the rna level of col27a1 was detected by linkedomics httplinkedomicsindeed mir4553pcatenindepletioninduced decrease of hsf1 mrna m6a modification fig 5c and additional file figs5f meanwhilethe interaction of mettl3 with hsf1 mrna wasinhibitorrescuedabrogated by depleting catenin fig 5d and additionalfile figs5g accompanied by the increased interactionof mir4553p to hsf1 mrna fig 5e and upregulationof mature fig 5f and additionalfile figs5h 0csong molecular cancer page of precursor additional file figs5i and primary mir4553p fig 5g and additional file figs5j in contrastwhen catenin was upregulated by overexpression or licltreatment both mature mir4553p fig 5f and additionalfile figs5h and primary mir4553p fig 5g and additional file figs5j were downregulatedprimary mir4553p was derived from a premirna hairpin encoded in intron of the collagen gene col27a1 additional file figs5k actually col27a1 expression was significantly correlated with the expression ofmir455 httplinkedomics fig 5h consistent withthis we observed col27a1 mrna levels were increasedupon catenin depletion while decreased after licl treatment fig 5i and additional file figs5l moreover catenintcf7l2 complex could interact with the promoterof col27a1 while the pair of primers negativechipprimer at a position far away from the promoter region couldnot enrich col27a1 fig 5j and additionalfile figs5m meanwhile the protein expression of cateninwas negatively correlated with rna level of col27a1httplinkedomics fig 5k these results indicatedthat the transcription of col27a1 was inhibited by wntcatenin signalingleading to decreased biogenesis ofmir4553p therefore catenin facilitates the shift frommir4553p binding to m6a modification in hsf1 mrnaby suppressing mir4553p expression eventually promoting hsf1 protein translationboth genetic and chemical inhibition of hsf1 attenuatecolorectal carcinogenesis in micein light of these in vitro findings we further exploredthe relevance of hsf1 to colorectal carcinogenesis inapcmin and apcmin hsf1 mice since the interaction of mouse mir4553p and mouse hsf1 mrnaseems to be well conserved mouse mir4553p seed sequence cagucca the binding site in mouse hsf1mrna ²utr tggactg the expression of hsf1 andits downstream target gls1 were increased whilemir4553p expression was reduced in intestine tissuesfrom apcmin mice compared with normal c57bl6mice fig 6a and b after fed with highfat diet for months these apcmin mice developed multiple tumorsin the intestine fig 6c however both the size andnumber of tumors were significantly reduced in apcminmice treated with a chemical inhibitor of hsf1 knk437and apcmin hsf1 mice fig 6d accompanied bythe downregulation of hsf1 targets fig 6e all of theseresults confirmed that hsf1 is a novel downstream target of wntcatenin signaling important to promotecrc developmentdiscussiongenetic changes in components of wntcatenin signaling such as deletion of the apc gene and ctnnb1mutations have been frequently detected in many typesof human cancers all of these muta | 0 |
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14 cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classiï¬ed into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in speciï¬c chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22 ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and ï¬broblastgrowth factor receptor FGFR gene fusions30 Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients beneï¬t from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classiï¬cation andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aï¬atoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver ï¬ukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difï¬culty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespeciï¬c survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Paciï¬c Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11 Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14 Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is aï¬rm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was ï¬rst described in cHCCICC was not systematicallydescribed until when it was classiï¬ed into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly deï¬ned areas type C mixedtype presents as intimate association without clear boundaries18 In another classiï¬cation system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identiï¬able intermediatetransition between HCC and ICC type III ï¬brolamellar tumors resemblesthe ï¬brolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classiï¬cationis commonly used in which cHCCICC is classiï¬ed into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a uniï¬ed classiï¬cation system greatly adds to the difï¬culty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdeï¬ned cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identiï¬ed stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA ampliï¬cations are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in of cases Recently some oncogenic genes were identiï¬ed in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identiï¬ed as a driver ofHCC45 VEGFA and CCND1FGF19 have also identiï¬ed in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identiï¬ed For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22 and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately of cases22 Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30 Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in of ICC cases32 Fusions ampliï¬cations translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC and ICC Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate ï¬lament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difï¬cult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166 Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efï¬cacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L ï¬rstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5ï¬uorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71 This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current ï¬rstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onï¬rstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand ï¬uoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the ï¬rstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe beneï¬t of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] conï¬dence intervalPFS months versus months p This effectiveness wasconï¬rmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the ï¬rstlinetreatment for advanced cholangiocarcinoma[CI] and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe ï¬rstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efï¬cacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efï¬cacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased beneï¬t for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095 Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI months CI months CI and months CI respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as ï¬rst line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir ï¬rstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the ï¬rst US Food and Drug Administration FDAapproved ï¬rstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPaciï¬c have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the ï¬rst generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have beneï¬tedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1 FGFR1 PDGFR RET and KIT In August theFDA approved lenvatinib for ï¬rstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the ï¬rstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modiï¬cations Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can speciï¬cally inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPaciï¬cREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further conï¬rmed the efï¬cacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival beneï¬t observed across all age subgroups and atolerable safety proï¬le supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety proï¬le apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic proï¬le and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a ï¬rstinhuman phase I study NCT02508467 of ï¬sogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123 Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identiï¬ed in recent years is the inhibitor of the ï¬broblast growth factorFGF signaling pathway which consists of members labeled FGF1FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1 FGF signals regulate cell proliferation in which FGFR2fusions occurred in of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the ï¬rst and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on ï¬ndings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may beneï¬t from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPï¬zeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial Identiï¬erTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi | 2 |
prevalence of pathogenic variants in DnA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of earlyonset renal cancerTiffiney a0R a0Hartman12 a0Elena a0V a0Demidova345 a0Randy a0W a0Lesh6 a0Lily a0Hoang7 a0Marcy Richardson7 a0Andrea a0Forman8 a0Lisa a0Kessler1 a0Virginia a0Speare7 a0Erica a0A a0Golemis4 a0Michael a0J a0Hall38 a0Mary a0B a0Daly38 Sanjeevani Arora3Pathogenic a0variants a0PVs a0in a0multiple a0genes a0are a0known a0to a0increase a0the a0risk a0of a0earlyonset a0renal a0cancer a0eoRC a0However a0many a0eoRC a0patients a0lack a0PVs a0in a0RCspecific a0genes a0thus a0their a0genetic a0risk a0remains a0undefined a0Here a0we a0determine a0if a0PVs a0in a0DNA a0damage a0response a0and a0repair a0DDRR a0genes a0are a0enriched a0in a0eoRC a0patients a0undergoing a0cancer a0risk a0assessment a0Retrospective a0review a0of a0deidentified a0results a0from a0 a0eoRC a0patients a0undergoing a0testing a0with a0a a0multigene a0panel a0for a0a a0variety a0of a0indications a0by a0Ambry a0Genetics a0PVs a0in a0cancerrisk a0genes a0were a0identified a0in a0 a0of a0patientswith a0 a0in a0RCspecific a0and a0 a0in a0DDRR a0genes a0DDRR a0gene a0PVs a0were a0most a0commonly a0identified a0in a0CHEK2 a0BRCA1 BRCA2 and ATM a0Among a0the a0 a0of a0patients a0with a0a a0BRCA1 or BRCA2 a0PV a0 a0 a0reported a0a a0personal a0history a0of a0hereditary a0breast a0or a0ovarianassociated a0cancer a0No a0association a0between a0age a0of a0RC a0diagnosis a0and a0prevalence a0of a0PVs a0in a0RCspecific a0or a0DDRR a0genes a0was a0observed a0Additionally a0 a0patients a0reported a0at a0least a0one a0additional a0cancer a0breast a0cancer a0being a0the a0most a0common a0 a0of a0females a0 a0of a0males a0Multigene a0testing a0including a0DDRR a0genes a0may a0provide a0a a0more a0comprehensive a0risk a0assessment a0in a0eoRC a0patients a0Further a0validation a0is a0needed a0to a0characterize a0the a0association a0with a0eoRCRenal cancer RC often develops with no signs or symptoms and is referred to as the silent disease While factors including smoking environment obesity and race have been linked to increased risk of RC inherited factors are the most wellvalidated source of increased risk2 Hereditary RC syndromes typically associated with earlyonset disease and a clinically significant family history of cancer result from germline pathogenic variants PV in highpenetrance RCspecific genes including VHL MET FLCN TSC1 TSC2 FH SDH PTEN and BAP15 A previous report of an earlyonset RC eoRC cohort screened with an RCspecific panel found of individuals had a PV in an RCspecific gene7 However for most eoRC patients a PV in an RCspecific gene is not identified leaving many eoRC genetically undefined Thus there is a need to identify additional genes related to eoRC risk Currently there are no National Comprehensive Cancer Network NCCN guidelines for detection prevention or risk reduction in individuals who present with an eoRC but lack a PV in a defined RCspecific gene81Arcadia University Glenside PA USA 2Cancer Biology Program Fox Chase Cancer Center Philadelphia PA USA 3Cancer Prevention and Control Program Fox Chase Cancer Center Cottman Avenue Philadelphia PA USA 4Molecular Therapeutics Program Fox Chase Cancer Center Philadelphia PA USA 5Kazan Federal University Kazan Russian Federation 6Geisinger Commonwealth School of Medicine Scranton PA USA 7Ambry Genetics Konica Minolta Aliso Viejo CA USA 8Department of Clinical Genetics Fox Chase Cancer Center Philadelphia PA USA email SanjeevaniArorafccceduScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cDNA damage response and repair genes DDRR play an important role in maintaining genome integrity and when mutated in the germline can increase cancer risk for several types of cancers including breast colorectal ovarian and others9 Although PVs in DDRR genes are associated with increased risk of a variety of cancer types they are not typically considered risk factors for RC However germline PVs in some DDRR genes have been observed in RC including PVs in the DNA mismatch repair Lynch syndrome genes MSH2 and MLH1 in renal urothelial carcinoma and PVs in CHEK2 in advanced renal cell carcinoma10 To address the hypothesis that PVs in additional DDRR genes may contribute to the missing heritability of eoRC we analyzed germline sequencing data from a cohort of individuals with RCMaterials and methodsAmbry a0Genetics a0eoRC a0study a0cohort a0and a0variant a0determination a0 Deidentified data were requested from RC patients that were tested by Ambry Genetics Konica Minolta Aliso Viejo California using germline cancer testing panels Ambry samples were selected for patients with RC and deidentified data was obtained for all RC patients tested with multigene cancer panels n a0years at diagnosis specimens collected between July December All genetic test results from germline testing of individuals diagnosed with RC at during this time period were used in this studyThere is currently no standard definition specifying the age when RC is considered earlyonset Different models have been used to determine a specific age as a trigger for germline testing in patients with RC who lack family history of RC including ages or a0years For this study we selected individuals a0years or younger as the cutoff for our cohort which is substantially below the median age of RC diagnosis of a0years in the general population as reported in SEER2223 but considerably older than other suggested cutoffs We did so because the main hypothesis of the study was that PVs in DDRR genes might be responsible for increased risk of RC Variants in multiple DDRR genes are associated with earlyonset colorectal cancer2425 which typically manifests in patients at a0years or younger We considered that PVs in DDRR genes were most likely to impact repair of DNA damage induced during cell replication leading to genetic instability and cancer given renal cells turn over much less frequently than colon cells we hypothesized that it may take longer for cancers associated with PVs in DDRR genes to manifest in RC causing us to select a cutoff of a0years old for assessmentDeidentified data included family history of cancer genetic test results personal history of cancers apart from RC presence of multifocal tumors and RCsubtypestage The RC patients had been tested with CancerNext versions and CancerNextExpanded versions and Table a0S1 Deidentified patient information was analyzed for genetic test results and personal and family medical histories Classification of variants by Ambry Genetics is based on ACMG recommendations for standards for interpretation and reporting of sequence variations These variants are also regularly deposited in ClinVar by Ambry Genetics Variant classification was updated through March for all data Gene variants were classified as pathogenic variant PVsee below for criteria variant of uncertain significance VUS or inconclusive or negativeindeterminate Ambry Genetics follows strict criteria when classifying variants as PV Variant Likely Pathogenic VLP VUS Variant Likely Benign VLB and Benign for details see wwwambry gencomclini cianourscien tific excel lence varia ntclass ifica tion Variants reported as PV and VLPs were grouped as PVs All test results were used for this study The analysis of VUS which currently lack clinical significance was beyond the scope of this study Given updating of test panels by Ambry Genetics not all patients were tested for all genes Individuals were provided different versions of the panel over the course of the study see below and also see Table a0S1Any deidentified personal or family history information including sex ethnicityrace age of cancer diagnosis tumor histology history of additional personal cancer and history of family cancer and types was reported first as summarized data and later as deidentified individual case reports For analysis comparing outcomes for RCspecific genes versus genes not typically associated with RC we focused our statistical comparison on only those individuals who had CancerNext Expanded panel version testing which analyzes all genes including the RCspecific genes individuals who had the CancerNext Expanded version test were used for this statistical comparison For additional statistical test comparisons that analyzed the correlations between specific genes and categories such as tumor pathology or age any individual who had been tested for that specific gene was includedThe Western IRB issued a regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus the requirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB provided study oversight and approval protocol number Ambry Genetics provided deidentified patient medical and family history where available and genetic results for the patients All methods were performed in accordance with the relevant guidelines and regulation of the approved studyGenetic a0analysis a0with a0Ambry a0CancerNext a0and a0CancerNext a0Expanded a0panels a0Individuals were provided different versions of the panel by their healthcare provider see Table a0S1 The number of genes in the panels ranged from the smallest CancerNext panel Version which include genes APC ATM BARD1 BRIP1 BMPR1A CDH1 CHEK2 EPCAM MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 PTEN RAD50 RAD51C SMAD4 STK11 TP53 to the largest CancerNext Expanded Version panel which contained genes APC ATM BAP1 BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM FH FLCN GREM1 MAX MEN1 MET MITF MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE PTEN RAD50 RAD51C RAD51D RET SDHA SDHAF2 SDHB SDHC SDHD SMAD4 SMARCA4 STK11 TMEM127 TP53 TSC1 TSC2 VHL The DDRRs identified in germline testing of this cohort are bolded26Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAmbry Genetics sequenced genomic DNA that was obtained from patient blood or saliva samples DNA was evaluated by next generation sequencing NGS of all coding sequences and ± bases into the ² and ² ends of flanking introns and untranslated regions In addition sequencing of the promoter region was performed for the following genes PTEN c to c MLH1 c to c and MSH2 c to c Additional Sanger sequencing was performed for any regions missing or with insufficient depth of coverage for reliable heterozygous variant detection and on potentially homozygous variants variants in regions with complicated pseudogene interference and when variant calls did not meet allele frequency quality thresholds Additional details on specific testing methods are available at wwwambry gencomclini ciangenet ictesti ng28oncol ogycance rnext expan dedControl a0population a0in a0ExAc a0and a0gnomAD a0 To compare the frequency of DDRR gene PVs found in the study to that in the general population our results were compared to the Exome Aggregation Consortium ExAc dataset of largely unrelated whole exome sequencing results and to the Genome Aggregation database gnomAD dataset consisting of exomes and genomes2728 These datasets are the most commonly used genomic data at the populationlevelClinVar a0analysis a0 ClinVar wwwncbinlmnihgovclinv ar a database of medically relevant gene variants was used to investigate all PVs in this study retrieved on February PVs that were not reported in ClinVar were noted as not reported Associated conditions for each PV were categorized into hereditary cancer predisposing syndromes conditions related to renal cancer and any other conditions To further elucidate any PVs related to renal cancer the search term renal cancer was queried and the results were noted as associated with ClinVar search term Renal CancerStatistical a0 analysis a0 To identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis genes were combined into pathwaysgroups of interest histologys were grouped and cancer types were grouped Each individual was categorized as having a variant in one of the genes within the group or no variant in the group Gene categories were used for comparison of RC diagnosis with a DDRR or an RCspecific geneWe also tested the hypothesis that different gene groups are associated with age at RC diagnosis We used the median age of RC diagnosis in the study cohort a0years and studied PVs in patients a0years or ¥ a0years of age To test the association between the presence of PVs and age of RC diagnosis twosided Fishers exact tests were used and a0pvalues were considered significant Odds ratios OR were calculated to determine the odds that an outcome had occurred given a particular variant compared to the odds of the outcome occurring in the absence of that variant in the population tested Finally we queried the SEER database for patients under a0years old with RC to compare the distribution of their clinical characteristics where available to those in our study cohort22Due to the evolving nature of the panels during the course of this study each version included a different total number of genes and analysis of each gene is based on the number of individuals whose test included that gene Table a0S1 Only data from individuals was considered for comparison of individuals with RCspecific genes compared to those with variants in genes not typically associated with RC as the other individuals did not have all genes analyzed For statistical comparisons analyzing correlations between specific genes with various characteristics all individuals who had been tested for that specific gene were includedTo identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis RCspecific genes other cancerassociated genes and DDRR genes were combined into groups and histologies were grouped The categories for comparison of PVs and patient characteristics are as follows Known RC genes BAP1 FH FLCN MEN1 MET MITF PTEN SDHA SDHAF2 SDHB SDHC SDHD TSC1 TSC2 and VHL versus genes not typically associated with RC APC ATM BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM GREM1 MAX MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D RET SMAD4 SMARCA4 STK11 TEMEM127 TP53 versus DDRR genes alone ATM BARD1 BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D Histology categories combined from the original categories Chromophobe Papillary renal Clear cell Wilms Renal cell likely clear cell but cannot be confirmed Unknown Mixed papillary [clear cell papillary type papillary renalchromophobe renal and sarcomatoidpapillaryclear cell] Mixed chromophobe [chromophobeoncocytoma chromophoberenal cell clear cellchromophobe and clear celloncocytomachromophobe] Oncocytoma Mixed oncocytoma [clear celloncocytoma oncocytomacollecting duct and renal celloncocytoma] and Others [included clear cellsarcomatoid collecting duct mixed epithelial and stromal mucinous tubular and spindle cell multilocular cystic renal neuroendocrine renal cellWilms renal cortical sarcomatoid transitional urothelial and urothelial transitional] Transitional urothelial urothelial and papillary transitional cases were not included in the analysis for counts of pathogenic variants Renal oncocytomas mixed epithelial and stromal tumors are considered benign tumors and were not included in the analysis for counts of pathogenic variants Study a0approval a0 The Western IRB issued a a0regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus a0the Scientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0crequirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB a0provided study oversight and approval protocol number Ambry Genetics a0provided deidentified results for the study All methods were performed in accordance with the relevant guidelines and regulation of the approved studyResultsPatient a0characteristics a0 We first benchmarked the eoRC study cohort to the reported incidence of RC in SEER data for the general US population to provide context In the study cohort of cases were between a0years of age and median age of diagnosis was a0years As expected a higher percentage of RC cases were diagnosed between a0years of age as compared to patients diagnosed with RC in the general US population SEER versus Fig a01A The study cohort was female and male Fig a01B Table a0 versus female and male for the general US population prevalence of RC diagnosed Fig a01B Raceethnicities in study cohort were Caucasian African AmericanBlack Ashkenazi Jewish Hispanic other and unknown Table a0The tumor pathologies reported varied Fig a01C and Table a0 Clear cell constitutes of all RCs in SEER and was the most commonly reported histology in the eoRC cohort Renal cell not defined but likely to predominantly reflect clear cell was also common Fig a01C and Table a0 Papillary and chromophobe histology were each identified in of the individuals and respectively Other histologies were identified rarely but included Wilms tumor and oncocytoma For of patients the RC subtype was unknownHigh a0incidence a0of a0other a0cancers a0in a0study a0cohort a0 n of the cases in the study cohort reported at least one additional primary cancer Fig a01D Table a0 Table a0S2 Each of the primary cancer types is also represented at a higher level in the study cohort than in the general US population as reported by the SEER database Fig a01D For femalespecific cancers of females also had breast cancer in comparison to the breast cancer rate in women in the general population SEER Fig a01D and Table a0S2 The rate of additional primary cancer in the study cohort is much higher than the rate of each cancer type observed in SEER cases with eoRC Fig a01E Finally patients out of reported a family history of cancer and of these patients specifically reported at least one family member with RC Table a0Multigene a0cancer a0panel a0testing a0identifies a0PVs a0in a0DDRR a0genes a0in a0the a0study a0cohort a0 The most common gene with PVs identified in the eoRC patients was the DDRR gene CHEK2 Fig a02A Table a0S3 and S4 consistent with a recent report by Carlo et a0al16 Of patients with CHEK2 PVs n had a common highly damaging variant c1100delC pThr367Metfs that is known to be associated with an increased risk for breast prostate colorectal and thyroid cancers Table a0S434After CHEK2 PVs were most frequently observed in the DDRR genes BRCA2 ATM and BRCA1 Table a0S3 We compared the overall frequency of PVs in CHEK2 BRCA1 BRCA2 and ATM to the control population in ExAc and gnomAD representing individuals sequenced for diseasespecific and population genetic studies2728 Overall PVs in each of these genes were more common in the study cohort versus the control populations Fig a02BC Table a0S5A An outlier was the moderate risk CHEK2 c470TC p I157T PV38 identified in individuals in the study cohort which was higher in the controls gnomAD OR CI ExAc OR CI We compared the prevalence of all PVs in DDRR genes presented in Table a0S4 from cases to controls from gnomAD23 We found 48fold enrichment of PVs in DDRR genes in the study cohort versus the controls in gnomAD vs respectively Table a0S5B each DDRR gene was corrected for number of patients assessedCancer risk with MUTYH DDRR gene has only been defined for individuals with homozygous or compound heterozygous PVs but not for heterozygous carriers39 We identified individuals with MUTYH PVs out of which were heterozygous carriers and only was compound heterozygous Only the individual with compound heterozygous MUTYH PVs was counted in the full study cohort n Table a0S3 and Fig a02A Similar to MUTYH cancer risk from the FH RCspecific gene c1431_1433dupAAA pK477DUP variant is currently considered to be pathogenic only in the compound heterozygous or homozygous state40 We identified RC patients who were heterozygous carriers of this specific FH variant Tables a0S3 and S4The overall gene variation rate in the full study cohort n is presented in Table a0S3 The full study cohort was not tested for all genes The largest panel was tested in the subcohort of cases and consisted of genes which included RCspecific genes and othercancer associated genes including DDRR genes Table a0S1 Here of cases had PVs PVs were identified in one or more of the genes not typically associated with RC in cases n Table a0S6 versus n with a PV in RCspecific genes Fig a02D Table a0S6 Of the genes not typically associated with RC were in DDRR genes n or n Among the patients patients were found to have PVs in two genes One patient had PVs in two DDRR genes BRCA1 and MUTYH het and the other patient in a RCspecific gene and a DDRR gene SDHB and MUTYH het Table a0S4 There was no MUTYH or FH compound heterozygous or homozygous PV in the subcohort of casesDDRR a0genes a0PVs a0are a0similarly a0enriched a0in a0patients a0diagnosed a0with a0eoRC a0alone a0or a0with a0eoRC a0and a0other a0cancers a0Individuals who were tested for all genes n could be further separated into two subcohorts those with eoRC as their only diagnosis n and those with eoRC and one or more additional types of cancer n To test the hypothesis that DDRR gene PVs might be Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAiega yb ssongad iCsesac AgenrnrWilmsersothal cellnrenrnwonknunramebohpomchroebod hmixepomchroar cellncocytocleodncocytomixeoamalnpillarypillary read pmixeapKey A C D E FSEER cohort n97805Ambry cohort n844Bsesac FemaleMaleSEER cohortAmbry cohortDtear recnac brainstabrectalolorecmiaekuleamonelamnariavoaticcrenapstateproamoarcsyroidthetrialuterinemodneEsesac number of primary cancers reportedFigure a0 Patient characteristics A Age range of individuals diagnosed with RC a0years in SEER cohort compared to the study cohort n of the remaining individuals in the study were diagnosed a0years were diagnosed at a0years and were excluded from the calculations as their age was reported as a wide range of years B Percentage of males and females diagnosed with RC a0years in SEER compared to the study cohort n C The percentages of reported RC histology up to age a0years in the SEER data n compared to the study cohort n not all histological subtypes reported in SEER were reported in the study cohort D The percentage of cancer incidence at a0years in the general SEER population versus the study cohort The SEER data reflect individuals reporting the indicated cancer type not individuals with RC in addition to the indicated cancer type E Percentage of different primary cancers reported a0years in SEER n versus the study cohort n Less than not reported for figure clarityScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cCharacteristicSexMaleFemaleEthnicityAfrican AmericanAshkenazi JewishAsianCaucasianHispanicMiddle EasternMixed EthnicityNative AmericanOtherUnknownMedian age of testingHistologyChromophobeMixed chromophobeClear cellOncocytomaMixed oncocytomaPapillary renalMixed papillaryRenal cellWilmsOthersUnknownPersonal cancer historyRenal cancer onlyRenal cancer plus additional cancer typeFamily history of cancerYesNoNot reportedunknownFamily history of renal cancerYesNoTotalNumber of patients in Ambry study cohort Rate in general population from birth to age SEER of renal cancers of renal cancersnrnrnrnrnrnr a0years Table Demographics and clinical characteristics of RC patients in the Ambry Genetics study cohort Demographics and clinical characteristics of the RC cases in the study cohort were compared to those of RC from birth to age in the SEER data Personal and family history of cancer were reported for the cases in the study cohort For family history of renal cancers numbers include only those who reported on cancer history n nr not reported SEER data included types of renal cancer histologies not all were represented in dataset other based on other category from Ambry cohort Family histories as selfreported on the intake formmedical records and have not been validatedassociated with eoRC we first analyzed PVs in eoRC cases with no additional primary cancer diagnosis Among the patients who only presented with eoRC PVs were identified in of cases n Fig a02E which is approximately twice the reported frequency of PVs in RCspecific genes7 Among this n of PVs were in one of DDRR genes ATM BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A NBN PALB2 RAD51C n were in one of RCspecific genes BAP1 FLCN SDHB VHL and the remaining cases bore PVs in nonDDRR genes associated with cancers other than RC Fig a02ENext we performed similar analysis as described above for patients who presented with eoRC plus one or more additional cancers Among the patients who presented with eoRC and at least one additional cancer Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cPVs were identified in cases Fig a02F Among these of cases PVs in othercancer associated genes including DDRR genes were found in of cases n versus n of cases with PVs in RCspecific genes This population was also enriched for PVs in DDRR genes n ATM BRCA1 BRCA2 CHEK2 MSH6 PALB2 PMS2 versus PVs in RCspecific genes BAP1 FLCN MET MITF PTEN SDHB VHLOverall these data suggest that DDRR gene PVs are enriched similarly in individuals diagnosed with eoRC alone or eoRC plus at least one additional primary cancer but that the frequency of PVs in DDRR genes in either group exceeded that in the control populations tested gnomADExAc Fig a0 Table a0S5A The specific PVs identified were similar in frequency to those identified in the full patient cohort n with CHEK2 the most represented DDRR genes Fig a0 To gain additional insight into the prevalence of these PVs in cancer patients we surveyed ClinVar wwwncbinlmnihgovclinv ar and found that multiple PVs from this study Table a0S4 have been reported in hereditary cancer predisposing syndromes HCPS summarized in Table a0S7 HCPS reflects a pattern of cancers in a family characterized by earlier onset with individuals not necessarily having the same tumor andor having more than one primary tumor and having tumors that are more likely to be multicentricRC a0patients a0with a0BRCA1 or BRCA2 a0PVs a0 Notably of the eoRC cases had a BRCA2 PV and RC cases had a BRCA1 PV Table a0 Table a0S3 This included n Table a0 of the cases who presented with only eoRC Interestingly despite the fact that the cohort was female of the detected BRCA1 and BRCA2 PVs were in males Table a0 Of the RC cases with a BRCA1 or BRCA2 PV had an additional cancer associated with hereditary breast and ovarian cancer HBOC syndrome breast ovarian prostate pancreatic or melanoma had an additional nonHBOC cancer and presented with only eoRC Table a0 Family history was reported for cases and of those indicated that at least one family member had an HBOCassociated cancer Of those with a BRCA2 PV reported that at least one family member had RC Table a0No a0correlation a0between a0age a0of a0RC a0diagnosis a0and a0type a0of a0PV a0in a0RC a0 To determine if identification of specific classes of germline PV correlated with age of diagnosis in this cohort genes were divided into four broad overlapping categories all genes in the panel RCspecific genes nonRC genes including DDRR genes and DDRR genes see Methods The groups were compared to median age at first RC diagnosis of or ¥ a0years Given the invariable earlyonset of Wilms tumor the individuals with this diagnosis were excluded from the analysis Within this eoRC cohort there was no significant association between age at diagnosis of RC and the type of PV for any of the four broad categories above Fig a03ACorrelation a0of a0renal a0histologies a0with a0PVs a0in a0specific a0genes a0 Of the clear cell cases in this cohort had a PV of which were RCassociated PVs Similar findings were observed for the cases described as renal cell carcinoma had a PV of which were RCassociated DDRR gene PVs were found in of clear cell cases and in of renal cell cases Figure a03BC contrast the findings in clear cell and renal cell histology with the other nonclear cell histologiesDiscussionThis study for the first time demonstrates that PVs in multiple DDRR genes occur in patients with eoRC Importantly this study found that DDRR gene PVs were represented both in cases diagnosed with eoRC and additional cancers and also cases diagnosed with eoRC alone Comparison with a large control population indicated that germline PVs in DDRR genes were more common in this study cohort than in the control population although further studies are required to confirm this finding and estimate the penetrance of PVs in DDRR genes for eoRC We also found that germline testing using an RCspecific panel would have identified only of the RC cases with actionable PVs according to the NCCN recommended screening or management guidelines compared to the additional cases identified with the expanded panelsThe most common gene with PVs identified in the patients in this study was the DDRR gene CHEK2 This is consistent with recent reports by Carlo et a0al and Huszno et a0al1516 While evidence is mounting that CHEK2 PVs may increase risk for RC in this study we did not consider CHEK2 as a gene typically associated with RC as it is not currently included on RC panels and would fail to be included in testing in many cases In addition limitations of the previous studies and the analysis reported here together indicate that larger studies with appropriate controls are needed before confirming that CHEK2 indeed confers a risk for RCIdentification of germline DDRR gene PVs can have specific implications for the proband and the family For example of cases diagnosed with eoRC alone had PVs in BRCA1 or BRCA2 but not all of these cases had a family history strongly indicative of HBOC syndrome This is important because identification of a BRCA PV can potentially change medical management for instance PARP inhibitor therapy is effective in tumors with BRCA PVs including nonbreast tumors4142 Also screening and prevention of HBOCsyndrome cancers would likely be increased significantly in the proband and in family members found to have the same PV Further many of the specific PVs identified in this study have been annotated as relevant to various HCPS emphasizing their role in the development of multiple cancer types Our results support broader panel testing as a way to identify unexpected highpenetrant PVs in eoRC patients when there is a personal or family history of additional cancers especially an HBOCsyndrome cancerScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cA stinairav cnegohapt lan deifitneditot BKey A D E FDDRR genesother cancer associated genesrenal cancer associated genesMTADRABACRBACRBPIRBKEHCHLMHSMHSMAERMHYTUMNBNBLAPSMPCDARCPAARPMBANKDCFNPTPABNCLFTEMFTIMNETPAHDSBHDSLHVPathogenic DDRR variants in Ambry cohort vs ExAc populationC Pathogenic DDRR variants in Ambry cohort vs GnomAD populationKey B CATM BRCA1BRCA2CHEK229211GA3576GA8655dupT5712dupA68_69delAG2475delC7558CT9294CG7069_7070delCT3847_3848delGT2339CG4284dupT518delG4441GA4441CT470TC1 | 2 |
"We also evaluated chest CT findings to determine the involvement of emphysema. The percentage of the COPD group with involvement of emphysema in the chest CT findings was almost twice as high as that of the non-COPD group (38.8% vs 20.3% respectively). Patient characteristics among non-COPD and COPD patients All cases (n?=?270) Non-COPD (n?=?123) COPD (n?=?147) p value Cases 100 (270) 45.6 (123) 54.4 (147) 0.0001 # Age years a 70.1 (3988) 67.9 (3982) 71.9 (5187) 0.0001 # Sex male 73.7 (199) 62.6 (77) 83.0 (122) 0.0001 # History of smoking 78.9 (213) 65.9 (81) 89.8 (132) 0.0001 # COPD managed b 8.5 (23) 1.6 (2) 14.3 (21) 0.0001 # COPD-related systemic comorbidities 55.9 (151) 52.8 (65) 58.5 (86) 0.390 Diabetes 19.3 (52) 16.3 (20) 21.8 (32) 0.280 Ischemic cardiac disease 7.4 (20) 2.4 (3) 11.6 (17) 0.004 # Hypertension 38.1 (103) 37.4 (46) 38.8 (57) 0.900 Hyperlipidemia 11.9 (32) 8.1 (10) 15.0 (22) 0.092 n indicates number. aData are shown as mean (range). bindicates the patients who had been diagnosed as COPD before bronchoscopy. All other data are shown as% (numbers). #p?<?0.05. COPD: chronic obstructive pulmonary disease. Population of non-COPD and COPD in Japanese patients with lung cancer. Schematic presentation of the percentage of non-COPD (n?=?123) and COPD (n?=?147) among Japanese patients with lung cancer. Patients with COPD were classified by GOLD grade that is grade 1 (n?=?95) grade 2 (n?=?41) grade 3 (n?=?11) and grade 4 (n?=?0). Physical assessment variables among non-COPD and COPD patients All cases (n?=?270) Non-COPD (n?=?123) COPD (n?=?147) p value BMI (kg/m 2 ) a 22.1 (2.8) 22.0 (2.8) 22.2 (2.9) 0.749 spirometric variables %VC a 105.9 (20.6) 104.8 (20.8) 106.7 (20.5) 0.520 FEV1 (ml) a 2062 (610) 2302 (555) 1861 (583) 0.0001 # FEV1/FVC a 67.3 (12.6) 77.8 (6.3) 58.9 (10.3) 0.0001 # %FEV1 predicted a 98.2 (25.7) 109.4 (21.1) 88.9 (25.4) 0.0001 # %IC a 85.9 (19.5) 83.6 (17.8) 87.9 (20.6) 0.111 chest CT finding emphysema 30.4 (82) 20.3 (25) 38.8 (57) 0.001 # n indicates number. aData are shown as mean (SD). All other data are shown as% (numbers). #p?<?0.05. BMI: body mass index; VC: vital capacity; FEV1: forced expiratory volume in 1 second; FVC: forced vital capacity; IC: inspiratory capacity; GOLD: the Global Initiative for Chronic Obstructive Lung Disease. Association of COPD prevalence with lung cancer characteristics in Japanese patients undergoing bronchoscopy To evaluate the association of COPD with characteristics of lung cancer the pathological findings EGFR mutation status clinical staging and decision for thoracic surgery were compared between the COPD group and the non-COPD group (). Characteristics of lung cancer status among non-COPD and COPD patients All cases (n?=?270) Non-COPD (n?=?123) COPD (n?=?147) p value Pathology 0.0001 # Adenocarcinoma 53.7 (145) 69.9 (86) 40.1 (59) ## Sq 27.0 (73) 17.9 (22) 34.7 (52) ## NSCLC 10.4 (28) 4.9 (6) 15.0 (21) ## SCLC 6.7 (18) 5.7 (7) 7.5 (11) Large 2.2 (6) 1.6 (2) 2.7 (4) Clinical stage 0.046 # 1A 25.6 (69) 30.8 (38) 21.1 (31) 1B 13.7 (37) 13.0 (16) 13.6 (20) 2A 9.6 (26) 13.0 (16) 7.5 (11) 2B 7.8 (21) 9.8 (12) 6.1 (9) 3A 11.9 (32) 8.9 (11) 14.3 (21) 3B 5.6 (15) 4.1 (5) 6.8 (10) 4 17.0 (46) 16.3 (20) 17.7 (26) ND 8.9 (24) 4.1 (5) 12.9 (19) ## Thoracic surgery 0.0001 # Yes 138 (51.1) 64.2 (79) 40.1 (59) EGFR mutation status 0.001 # Yes 14.8 (40) 21.1 (26) 9.5 (14) ## No 55.2 (149) 59.3 (73) 51.7 (76) ND 30.0 (81) 19.5 (24) 38.8 (57) ## n indicates number. All data are shown as% (numbers). ND indicates not determined. #p?<?0.05. ##indicates a significant difference compared with the non-COPD group. COPD: chronic obstructive pulmonary disease; Sq: squamous cell carcinoma; NSCLC: non-small cell lung carcinoma; SCLC: small cell lung carcinoma; Large: large cell carcinoma; EGFR: epidermal growth factor receptor. Regarding pathologic findings the incidence of adenocarcinoma was significantly lower in the COPD group than in the non-COPD group. In the present study EGFR mutation was observed only in the patients with adenocarcinoma (40/145 cases; 27.6%). In contrast the number of cases in which EGFR mutation status was not determined was significantly higher in the COPD group than in the non-COPD group. Although determination of the clinical stage should be essential in order to propose the therapeutic options for lung cancer some cases in which clinical staging had not been completed were observed in the study population. The number of these cases was significantly higher in the COPD group than in the non-COPD group. In contrast the proportion of patients with each classification in the clinical staging did not differ between the two groups besides the cases in which the clinical staging was not determined. Among the total study population the number of thoracic surgeries performed was significantly lower in the COPD group than in the non-COPD group. Critical impact of the severity of airflow obstruction on the decision to propose thoracic surgery with curative intent To explore whether or not the severity of airflow obstruction might affect the decision to propose thoracic surgery with curative intent patients at stage 3B and 4 were excluded from the analysis because they were not eligible for thoracic surgery. In addition patients for whom the clinical staging had not been completed were also excluded. As a result we evaluated data from 185 patients with lung cancer at stage 1A to 3A who underwent spirometry and bronchoscopy. These patients were subdivided into those who underwent thoracic surgery (135 cases) and those who did not (50 cases). The characteristics and spirometric data for the patients with or without thoracic surgery are summarized in Tables 4 and 5. The characteristics of lung cancer among these groups are also shown in . Univariate analysis identified a significantly lower frequency of thoracic surgery among the patients of greater age and with more severe airway obstruction and advanced clinical staging. Univariate analysis also identified a significantly higher frequency of thoracic surgery among patients with adenocarcinoma. Finally all of the factors with a significant association in the univariate analysis were applied to the multivariate model to identify variables independently associated with the decision for thoracic surgery. Multivariate analysis identified more severe airway obstruction advanced clinical stagings and higher age as independent factors affecting the decision on thoracic surgery ()." | 1 |
the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second peoples hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second peoples hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at a0 °c for further experiments this research was approved by the ethics committee of lianyungang second peoples hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbeccos modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0 with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative realtime polymerase chain reaction qrtpcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit cck assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of à a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandualluciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenolchloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells à transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second peoples hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by students t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second peoples hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second peoples hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of à a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi | 0 |
"In we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC. KRAS EGFR Lung Cancer Radiation Med Phys Med Phys Medical Physics 0094-2405 0094-2405 American Association of Physicists in Medicine 24694124 3978354 020404MPH 10.1118/1.4867860 Radiation Therapy Physics A block matching-based registration algorithm for localization of locally advanced lung tumors Robertson Scott P. Weiss Elisabeth Hugo Geoffrey D. a) Department of Radiation Oncology Virginia Commonwealth University Richmond Virginia 23298 a) Author to whom correspondence should be addressed. Electronic mail: gdhugovcu.edu; Telephone: 804-628-3457; Fax: 804-628-0271. 4 2014 13 3 2014 41 4 041704 24 6 2013 17 2 2014 20 2 2014 Copyright 2014 American Association of Physicists in Medicine 2014 American Association of Physicists in Medicine Purpose: To implement and evaluate a block matching-based registration (BMR) algorithm for locally advanced lung tumor localization during image-guided radiotherapy. Methods: Small (1 cm3) nonoverlapping image subvolumes (blocks) were automatically identified on the planning image to cover the tumor surface using a measure of the local intensity gradient. Blocks were independently and automatically registered to the on-treatment image using a rigid transform. To improve speed and robustness registrations were performed iteratively from coarse to fine image resolution. At each resolution all block displacements having a near-maximum similarity score were stored. From this list a single displacement vector for each block was iteratively selected which maximized the consistency of displacement vectors across immediately neighboring blocks. These selected displacements were regularized using a median filter before proceeding to registrations at finer image resolutions. After evaluating all image resolutions the global rigid transform of the on-treatment image was computed using a Procrustes analysis providing the couch shift for patient setup correction. This algorithm was evaluated for 18 locally advanced lung cancer patients each with 47 weekly on-treatment computed tomography scans having physician-delineated gross tumor volumes. Volume overlap (VO) and border displacement errors (BDE) were calculated relative to the nominal physician-identified targets to establish residual error after registration. Results: Implementation of multiresolution registration improved block matching accuracy by 39% compared to registration using only the full resolution images. By also considering multiple potential displacements per block initial errors were reduced by 65%. Using the final implementation of the BMR algorithm VO was significantly improved from 77% ± 21% (range: 0%100%) in the initial bony alignment to 91% ± 8% (range: 56%100%; p < 0.001). Left-right anterior-posterior and superior-inferior systematic BDE were 3.22.4 and 4.4 mm respectively with random BDE of 2.42.1 and 2.7 mm. Margins required to include both localization and delineation uncertainties ranged from 5.0 to 11.7 mm an average of 40% less than required for bony alignment. Conclusions: BMR is a promising approach for automatic lung tumor localization. Further evaluation is warranted to assess the accuracy and robustness of BMR against other potential localization strategies. nonsmall-cell lung cancer image registration image-guided radiation therapy CCC information 0094-2405/2014/41(4)/041704/11/$30.00 sisac 0094-2405()41:4L.041704;1 edcode 13-932R2 aipkey 1.4867860 spin KART 2012 8757Q- 8757nj 8757cf 8719xj 8753Jw US 10 cancer computerised tomography image matching image registration image resolution lung median filters medical image processing radiation therapy tumours ipc-code A61B6/03 Computerised tomographs ipc-code A61N5/00 Radiation therapy ipc-code G01N33/48 Biological material e.g. blood urine; Haemocytometers ipc-code G06F19/00 Digital computing or data processing equipment or methods specially adapted for specific applications ipc-code G06T Image data processing or generation in general PLoS One one 1932-6203 Public Library of Science San Francisco USA 24587153 3935965 PONE-D-13-45372 .0089961 Research Biology Developmental biology Morphogenesis Cell migration Model anisms Animal models Mouse Molecular cell biology Cell growth Medicine Oncology Basic cancer research Metastasis Cancers and neoplasms Gastrointestinal tumors Hepatocellular carcinoma Luteoloside Suppresses Proliferation and Metastasis of Hepatocellular Carcinoma Cells by Inhibition of NLRP3 Inflammasome Luteoloside Suppresses HCC Carcinogenesis Fan Shao-hua 1 Wang Yan-yan 2 Lu Jun 1 Zheng Yuan-lin 1 * Wu Dong-mei 1 Li Meng-qiu 1 Hu Bin 1 Zhang Zi-feng 1 Cheng Wei 3 Shan Qun 1 1 Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province School of Life Science Jiangsu Normal University Xuzhou Jiangsu China 2 Department of Function Examination The First People's Hospital of Xuzhou Jiangsu China 3 School of Environment and Spatial Informatics China University of Mining and Technology Xuzhou Jiangsu China Sarkar Devanand Editor Virginia Commonwealth University United States of America * E-mail: ylzhengjsnu.edu.cn Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: SF JL YZ. Performed the experiments: SF YW DW ML WC. Analyzed the data: SF YW JL. Contributed reagents/materials/analysis tools: YZ BH ZZ QS. Wrote the paper: SF YW JL YZ. 2014 26 2 2014 9 2 e89961 13 10 2013 25 1 2014 2014 Fan et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. The inflammasome is a multi-protein complex which when activated regulates caspase-1 activation and IL-1? secretion. Inflammasome activation is mediated by NLR proteins that respond to stimuli. Among NLRs NLRP3 senses the widest array of stimuli. NLRP3 inflammasome plays an important role in the development of many cancer types. However Whether NLRP3 inflammasome plays an important role in the process of hepatocellular carcinoma (HCC) is still unknown. Here the anticancer effect of luteoloside a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla against HCC cells and the underlying mechanisms were investigated. Luteoloside significantly inhibited the proliferation of HCC cells in vitro and in vivo. Live-cell imaging and transwell assays showed that the migration and invasive capacities of HCC cells which were treated with luteoloside were significantly inhibited compared with the control cells. The inhibitory effect of luteoloside on metastasis was also observed in vivo in male BALB/c-nu/nu mouse lung metastasis model. Further studies showed that luteoloside could significantly reduce the intracellular reactive oxygen species (ROS) accumulation. The decreased levels of ROS induced by luteoloside was accompanied by decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1. Inactivation of caspase-1 by luteoloside resulted in inhibition of IL-1?. " | 1 |
"objective endoscopic full thickness resection eftr has shown efficacy and safety in the colorectum the aim of this analysis was to investigate whether eftr is cost effective in comparison with surgical and endoscopic treatment alternativesdesign real data from the study cohort of the prospective single arm wall resect study were used a simulated comparison arm was created based on a survey that included suggested treatment alternatives to eftr of the respective lesions treatment costs and reimbursement were calculated in euro according to the coding rules of and eftr r0 resection rate was used as a measure of effectiveness to assess cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were determined calculations were made both from the perspective of the care provider as well as of the payerresults the cost per case was ¬ for the eftr group ¬ for the standard endoscopic resection ser group ¬ for the surgical resection group and ¬ for the pooled alternative treatment to eftr from the perspective of the care provider the acer mean cost per r0 resection was ¬ for eftr ¬ for ser ¬ for surgical treatment and ¬ for all pooled and weighted alternatives to eftr the icer additional cost per r0 resection compared with eftr was ¬ for ser ¬ for surgical resection and ¬ for the pooled rate of alternatives results from the perspective of the payer were similar eftr is cost effective in comparison with surgical and endoscopic treatment alternatives in the colorectumintroductioncolorectal cancer is the third most common type of cancer and the second most common cause of cancer related deaths worldwide1 screening programmes for early detection of premalignant and malignant lesions led summary boxwhat is already known about this subject º endoscopic full thickness resection eftr has shown clinical efficacy and safety in difficult to treat lesions in the colorectum º the cost of the full thickness resection device is higher than the cost of standard endoscopic resection ser devices but lower than surgical devices º cost effectiveness analyses on treatment with eftr compared with treatment alternatives do not existwhat are the new findings º eftr leads to an almost reduction in cost per r0 resection average cost effectiveness ratio compared with surgery º to achieve an additional r0 resection by surgical treatment compared with eftr incremental cost effectiveness ratio an additional cost of ¬ is necessary º these findings are consistent both from the perspective of the care provider as well as the payerhow might it impact on clinical practice in the foreseeable future º in terms of cost effectiveness eftr should be considered first before patients with difficult to treat lesions in the colorectum are sent to surgical treatmentto a significant reduction in cancer related mortality2 with more intense screening more lesions are detected which automatically creates the need for removal standard endoscopic resections ser such as endoscopic mucosal resection emr and endoscopic submucosal dissection esd are well established and sufficient for the vast majority of lesions however ser of non lifting lesions and lesions located at difficult anatomical to cite kuellmer a0a behn a0j beyna a0t et a0al endoscopic full thickness resection and its treatment alternatives in difficult to treat lesions of the lower gastrointestinal tract a cost effectiveness analysis bmj open gastro 20207e000449 101136bmjgast2020000449received may revised july accepted july authors or their employers re use permitted under cc by nc no commercial re use see rights and permissions published by bmjfor numbered affiliations see end of correspondence toarthur schmidt arthur schmidt uniklinik freiburg dekuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access locations eg appendiceal orifice is associated with increased complication rates or incomplete resection3 these types of lesions are therefore often referred to surgery which is associated with significant morbidity and mortality and higher costs6 given the high number of polypectomies performed worldwide this is not only an issue of morbidity and mortality but also a huge economic challengethe efficacy and safety of endoscopic full thickness resection eftr of non lifting and other difficult to treat lesions have been demonstrated in multiple retrospective studies and in one prospective study7 the cost of the device is markedly higher than ser devices but lower than surgical treatment the aim of the present analysis was to evaluate whether eftr is cost effective in comparison with ser as well as surgical treatmentmethodsstudy populationto calculate the cost of eftr we analysed the study cohort of the wall resect trial nct02362126 in this single arm multicentre prospective study patients with difficult to treat adenomas eg non lifting andor challenging anatomical location early adenocarcinomas and subepithelial tumours in the colorectum were treated with eftr the primary endpoints of the study en bloc and r0 resection rate were achieved in and respectively7 written informed consent was obtained from each patient included in the studysimulation second study arm based on a survey of endoscopistswith the wall resect study being single armed a second study arm was created based on treatment simulation in order to compare different treatment modalities a case report form crf was created and sent to each participating centre of the wall resect trial endoscopists at the respective location reviewed the endoscopic images and their case relevant data and decided which treatment modality they would have chosen if eftr were not available treatment alternatives included ser such as emr thermal methods and esd as well as surgical resection the crf was filled out in a pseudonymised fashioninformed consent had already been obtained within the wall resect studydetermination of case costs and reimbursementa certified online it tool the diagnosis related group drg web grouper was used to determine the reimbursement rate for each patient http drg uni muenster de index php therefore the code of the international classification of diseases icd10 and the specific code for the procedure performed operationen und prozedurenschl¼ssel ops code in each patient in both groups were put into the web grouper together with the predefined mean length of hospital stay mittlere grenzverweildauer the drg which accounts for reimbursement was calculatedin the comparison arm the drg codes of were used because this was the year the wall resect study was performed for the eftr arm the drg codes of were used as reimbursement for eftr was increased that yearto calculate the cost per case another certified online it tool g drg report browser was used www g drg de g drg system_ abschlussbericht_ zur_ weiterentwicklung_ des_ g drg systems_ und_ report_ browser this was done by filling in the respective drg icd10 and ops code into the browser the data of the g drg report browser derive from the data that were sent in to inek authority managing the german drg system by certified hospitals kalkulationshuser in grouping was performed following the rules of g drg version the main and secondary diagnoses are shown according to icd10 german modification gm version and the procedures according to ops version g drg report browser inek gmbhas reimbursement for the eftr group was taken from the cost per patient case would ideally also have been calculated from unfortunately these data will be first published by inek in to overcome this problem costs for eftr cases from the university of freiburg between and which were reported to inek were used for the analysiswith the cost of each patient case the mean cost for each treatment modality emr esd laparoscopic surgery transanal endoscopic microsurgery tem eftr could be calculated in the next step the mean cost for each treatment path ser surgical treatment and casemix alternative was determined this was done in the following fashion for ser the mean costs of emr and esd were used for calculation of the surgical treatment laparoscopic surgery and tem were taken together the mean costs of endoscopic and surgical treatments were subsumed as the casemix alternativedetermination of effectivenessthe r0 resection rate was defined as the efficacy parameter to determine cost effectiveness the r0 rate of eftr in the wall resect trial was to determine the efficacy of the therapeutic alternatives to eftr a selective literature review was performed in pubmed and cochrane databases identifying the largest studies comparing resection techniques and r0 rates the respective rates regarding ser found in the literature were for emr and for esd9 for the surgical oncological resection treatment as the gold standard a r0 resection rate was assumed for the tem a rate of had been reported10in order to compare all ser methods emresd all surgical resection methods laparoscopic surgerytem and all alternative methods endoscopic and surgical kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0ctable alternative treatment strategies to eftr with their respective efficacy based on literature review and calculationtreatmentefficacy n n180surgical oncological resection laparoscopictememresdsurgical treatment laparoscopic and temser emresdcasemix alternative assumed arezzo et al fujiya et al arezzo et al calculated calculated calculatedthe combined effectiveness of surgical treatment ser and casemix alternative was calculated by multiplication of the number of patients in each modality eg emr cases for ser with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group overall efficacy of surgical treatment and casemix alternative was performed in the same mannereftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycasemix alternative with the eftr procedure a combined effectiveness of each treatment group was calculated this was done by multiplication of the number of patients in each modality eg emr cases with the respective r0 resection rate as the first step in the second step this result would be summed up to the result of the other modalities eg esdemr result for the ser methods and divided by the number of patients in this group of resection method eg patients in the ser group using this approach the overall efficacy in the ser group was calculated as overall efficacy of surgical treatment and casemix alternative was performed in the same manner and was calculated as and respectively the respective r0 rates are shown in table calculation of costeffectivenessfor assessment of cost effectiveness the average cost effectiveness ratio acer and the incremental cost effectiveness ratio icer were calculated acer expresses the mean costs for the investigated outcome11 in our study acer describes the mean costs per successful r0 resection in the different treatment modalitiesacer is calculated with the following computational formula acer mean costseffect open accessicer expresses the additional costs of a treatment alternative for improvement in the investigated outcome12 in our study these are the incremental costs for the alternative treatment to eftr required to achieve an r0 resectionicer is calculated with the following computational formula icer mean costs interventionmean costs controleffect intervention effect control the mean costs were the total costs of the respective treatment modality divided by the number of patients in each group for the calculation of cost effectiveness the ser methods emr and esd as well as the surgical resection methods laparoscopic resection and tem were taken together furthermore cost effectiveness was calculated for the casemix alternative to compare eftr with all alternativesresultscomparative study armendoscopist surveyfrom patients of the study cohort responses were included for further analysis in one patient the investigator recommended solely thermal ablation as alternative treatment of choice thus the primary endpoint r0 resection could not be evaluated from the remaining patients the endoscopists recommended surgical treatment in of of cases thereof of were laparoscopic resections and of tem in of of cases an endoscopic resection was proposed thereof of were emr and of were esdcosts from the perspective of the care providercosts per case were derived from the drg report browser which represent costs of the respective treatment alternative for the year the costs for the eftr treatment were derived from university hospital freiburg and represent the mean costs from years to mid2019 the mean cost for eftr was ¬ the cost per surgical treatment laparoscopic surgery and tem was ¬ and for ser ¬ all alternative treatment strategies casemix alternative op laparscopic surgery tem esd and emr were calculated as ¬ per case the results are shown in figure costs from the perspective of the thirdpartyaccording to the german drg system reimbursement for eftr is ¬ for surgical treatment ¬ was calculated the cost per case for ser is ¬ the cost for the casemix alternative is ¬ the results are shown in figure costeffectiveness analysis care provider viewpointaverage costeffectiveness ratiothe mean cost per r0 resection is ¬ in the eftr group and ¬ in the surgical group in the ser group the cost per r0 resection is ¬ in the casemix alternative group including all treatment kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access figure case costs ¬ for the different treatment modalities are shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryfigure incremental cost effectiveness ratio for the different treatment modalities compared with eftr is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgeryalternatives except eftr the mean cost per r0 resection is ¬ the results are shown in figure the casemix alternative ¬ the results are shown in figure incremental costeffectiveness ratioin comparison with eftr the incremental cost for an additional r0 resection is ¬ if ser is performed the cost for the surgical approach is ¬ and for figure average cost effectiveness ratio ¬ for the different treatment modalities is shown costs from the perspective of the third party payer reimbursement are shown in black while actual case costs from the perspective of the care provider are shown in grey surgery mean costs for tem and laparoscopic surgical oncological resection ser mean costs for esd and emr casemix mean costs for esd emr tem and laparoscopic surgery eftr endoscopic full thickness resection emr endoscopic mucosal resection esd endoscopic submucosal dissection ser standard endoscopic resection tem transanal endoscopic microsurgerycosteffectiveness analysis health insurance reimbursement viewpointaverage costeffectiveness ratiofrom the perspective of the health insurance the cost per r0 resection is ¬ in the eftr group in the ser group the cost is ¬ and in the surgical treatment group ¬ in the casemix alternative the cost per r0 resection is ¬ the results are shown in figure incremental costeffectiveness ratiothe icer of ser in comparison with eftr is ¬ the surgical approach costs an additional ¬ for the casemix alternative ¬ is necessary for an additional r0 resection the results are shown in figure discussionwith technical endoscopic progress patient care has constantly improved over the years however as with any technical innovation this is associated with higher costs therefore the efficacy of new methods and devices needs to be evaluated in relation to their costs13 to our knowledge this is the first cost effectiveness analysis cea for eftr our results demonstrate that eftr for difficult to treat lesions in the colorectum is cost effective in comparison with ser as well as surgical therapy furthermore the results are consistent when analysed from the perspective of the care provider as well as of the payerkuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0cfor our analysis a simulated control arm was created this was necessary as to date no randomised controlled trial rct investigating eftr versus alternative treatments has been published in our survey endoscopists proposed surgical treatment as the likely alternative to eftr in the majority of cases as opposed to ser via emr or esd all lesions within the wall resect trial were difficult to resect lesions eg non lifting adenomas exhibiting a high risk of perforation or incomplete resection when treated with ser therefore it may be surprising that ser was suggested in of cases however the suggestions were made by expert endoscopists who might have decided towards an advanced endoscopic procedure more generouslyregarding the costs for each treatment modality it was not surprising that the cost of eftr is above ser ¬ vs ¬ this is due to the cost of the device in germany ¬ plus value added tax however the cost of eftr was roughly one third of the cost of surgery ¬ vs ¬ this reflects the minimally invasive nature of eftr compared with laparoscopic or open surgical operationswhile costs for endoscopic resection and surgical therapy were taken from official and certified tools web grouper and drg report browser the factual costs of the eftr procedure for the year that are determined in a representative cross section of hospitals have not been published yet by inek the administrator of the drg system reimbursement of the procedure changed in thus these data should have been used for calculation to overcome this problem the mean case costs for eftr per case in our home institution university hospital of freiburg germany in the time between and were used as a surrogate in an economic analysis of the cost of eftr in germany presented at the annual conference of the german society for digestive diseases obtained from different endoscopic centres reported ¬ per case as this is only above our number and therefore in the same range our calculated ¬ seems to be a realistic number14in our analysis we chose the r0 rate as a means to detemine effectiveness as this is the most objective parameter to assess curative resection and treatment success furthermore the r0 rate can be compared with the treatment alternatives of eftr as high quality meta analyses and therapeutic success rates exist for those procedures10 the r0 rate for surgical colonic resection was assumed to be however the patient cohorts of these studies are not equal the wall resect study included only difficult to resect lesions mainly non lifting while the studies mentioned above included primarily treatment naive lesions larger studies on ser on non lifting lesions do not exist hence it is reasonable to assume that in these indications real r0 rates of ser would be lower and therefore cost effectiveness would be even worsefor measuring cost effectiveness acer and icer were determined the analysis was performed both open accessfrom the perspective of the care provider hospital as well as the reimbursement authority health insurance acer expresses the mean cost per r0 resection for both investigated perspectives our results reveal that costs are much lower for eftr compared with the surgical alternative although the effectiveness of the surgical approach in terms of radicality can be considered to be higher eftr is cost effective an r0 resection by eftr leads to nearly reduction in costs for the care provider ¬¬ and for the health insurance ¬¬ compared with ser eftr leads to marginally higher costs per r0 resection as explained above comparing eftr with ser has limitations as the investigated difficult lesions in the wall resect study are not well studied for emr and esd however in comparison with all treatment alternatives casemix alternative we calculated and reduction in costs similar to the surgical alternatives figure icer expresses the additional costs for an additional increase in the designated outcome in our analysis it expresses the additional costs that are necessary for an additional r0 resection as shown in figure all alternatives to eftr result in additional costs while ser results in a modest increase ¬ and ¬ additional ¬ and ¬ per r0 resection are required in the surgical group in the casemix alternative group additional costs were ¬ and ¬ respectivelyan absolute threshold at which an icer is thought to be cost effective does not exist16 in the literature the willingness to pay threshold ranges from to and is highly subjective to the investigated outcome and the healthcare system for which the cea is made17 for our analysis we assume that a more invasive treatment that produces at least ¬ more costs for an additional r0 resection cannot be regarded as being cost effectivefor our analysis we did not include costs of follow up endoscopies or further treatment arising from recurrency or from adverse events this was done due to the following reasons first reliable recurrence rates and long term follow up after eftr do not exist follow up in the wall resect trial was only weeks second the lesions of our patient cohort were heterogeneous including adenomas carcinomas and neuroendocrine tumours with different biological features and recurrent rates third treatment of recurrent lesions is not standardised and ranges from re eftr to snare polypectomy to removal with a biopsy forceps leading to highly variable costs fourth management of severe complications and consecutive morbidity differs in every patient and depends on severity of complication patients comorbidities and local expertise we do not have reliable data on costs for such treatment and a hypothetical model would have been highly speculative moreover in the wall resect trial of patients required consecutive surgery due to complications this rate is slightly kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0copen access higher but still grossly comparable with the complication rates of emr and esd on the other hand complications after surgical resection eg anastomotic leakage are much more frequent up to and usually lead to higher morbidity hence even if costs related to complications were added icer is still likely to favour eftr compared with the group of treatment alternativesit is difficult to compare our results with other ceas as this is the first one for this indication the only previous cea on ser compared emr and esd in laterally spreading lesions irrespective of location or lifting sign in most analyses as in the study by bahin and colleagues19 a decision tree model was created to compare different outcome scenarios after each treatment path was filled with probabilities of occurrence costs per predefined outcome were calculated a potential bias of this approach is that the data for the probabilities of occurrence which influence the costs most are taken totally or at least in part from different studies19 22this harbours the risk of resulting in a very heterogeneous study population with uncontrolled confounders this risk can be minimised by deriving data from rcts with well balanced patient cohorts as recently published17 for our analysis we used a different approach than a decision tree factual variables and outcome data derived from the only prospective study on eftr treatment and not from assumptions the simulation of the control arm had to be performed due to the lack of rcts in this setting the strength of our study is that the very same clinician who actually performed the respective eftr could review the different lesions and decide on a solid basis which treatment alternative he or she would have used instead of eftr in our view this approach reflects the clinical situation more precisely than a decision tree modelin most ceas the costs per quality adjusted life years are calculated and taken for healthcare decisions neither survival nor quality of life measurements were part of the wall resect trial in line with most of the recently published cea we calculated costs per defined outcome as the primary endpoint17 our study has several limitations first the comparison arm of the study is based on simulation so there is always a risk of a bias second our analysis is specific to the german healthcare system and may therefore not be fully comparable with different healthcare systems in the world third the estimated r0 rate for the ser methods is very low and likely due to the piecemeal resection in the respective study if efficacy would have been measured as freedom of recurrence efficacy would be higher as proven in the australian colonic endoscopic study24 nonetheless we used the published r0 rate because of the possibility to match this with the endpoint of the wall resect study furthermore as described above an endpoint such as freedom of recurrence cannot be determined reliably as such data do not exist for eftr fourth costs of complications and follow up were not included this is mainly due to lack of an rct and the short follow up period in an ideal cea all treatment related and hospital stay related costs would have been calculatedin our data indicate that eftr for difficult to treat lesions of the colorectum is cost effective compared with surgical and endoscopic treatment alternatives the results are consistent both from a care provider as well as from a third party payer perspective rcts and long term follow up are needed to further assess the cost effectiveness of eftrauthor affiliations1department of medicine ii medical center university of freiburg faculty of medicine university of freiburg freiburg germany2department of gastroenterology klinikum ludwigsburg ludwigsburg baden w¼rttemberg germany3department of gastroenterology evangelisches krankenhaus d¼sseldorf dusseldorf nordrhein westfalen germany4department of internal medicine and gastroenterology elisabeth hospital essen nordrhein westfalen germany5department of medicine ii interventional and experimental endoscopy inexen university hospital wurzburg wurzburg bayern germany6department of gastroenterology university hospital augsburg augsburg bayern germany7department of gastroenterology university hospital ulm ulm baden w¼rttemberg germany8department of gastroenterology klinikum dortmund dortmund nordrhein westfalen germany9department of gastroenterology helios klinikum krefeld krefeld nordrhein westfalen germany10department of gastroenterologyoncology klinikum sindelfingen b¶blingen sindelfingen baden w¼rttemberg germanycontributors as and kc invented and planned the present study and also the underlying wall resect study as assisted with data acquisition and analysis and revised the manuscript jb was responsible for data research and acquisition ak was responsible for data analysis and writing the manuscript kc tb bs am hm hn da mb ap mf tf mg and rt took part in the online survey to create the simulation comparison arm of the study furthermore they carefully read and revised the manuscriptfunding the authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorscompeting interests as and kc received lecture fees and study grants from ovesco endoscopy t¼bingen germany ak jb tb bs am hm hn da mb ap mf tf mg and rt have no conflicts of interest or financial ties to disclosepatient consent for publication not requiredethics approval the wall resect study was approved by the ethical board on january the study protocol conforms to the ethical guidelines of the declaration of helsinki as reflected in a prior approval by the institution's human research committee for the present study an additional approval by the institutional review board was not necessary since no additional personal data were collectedprovenance and peer review not commissioned externally peer revieweddata availability statement all data relevant to the study are included in the or uploaded as supplementary information data were derived from the wall resect trial nct02362126open access this is an open access distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idarmin a0kuellmer http orcid org kuellmer a0a et a0al bmj open gastro 20207e000449 101136bmjgast2020000449 0creferences who colorectal cancer fact sheet the global cancer observatory available http gco iarc fr today data factsheets cancers 10_ 8_ colorectum fact sheet pdf [accessed sep ] zauber ag winawer sj o'brien mj et a0al colonoscopic polypectomy and long term prevention of colorectal cancer deaths n engl j med hong sn byeon js lee b i et a0al prediction model and risk score for perforation in patients undergoing colorectal endoscopic submucosal dissection gastrointest endosc org mizushima t kato m iwanaga i et a0al technical difficulty according to location and risk factors for perforation in endoscopic submucosal dissection of colorectal tumors surg endosc agapov m dvoinikova e factors predicting clinical outcomes of endoscopic submucosal dissection in the rectum and sigmoid colon during the learning curve endosc int open 20142e235 baum p diers j lichthardt s et a0al sterblichkeit und komplikationen nach viszeralchirurgischen operationen dtsch arztebl int schmidt a beyna t schumacher b et a0al colonoscopic full thickness resection using an over the scope device a prospective multicentre study in various indications gut aepli p criblez d baumeler s et a0al endoscopic full | 0 |
" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncolli w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol lausen b schaumacher m maximally selected rank statistics biometricslausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p mantovani a allavena p sica a balkwill f cancerrelated inflammationnature giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncolli c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the | 0 |
inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf EbrahimzadehPustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon JayJiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factorκB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patientderived GBM cells cultured in 3D microwells were cotreated with BAY and TMZ or BAY and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reversephase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the cotreatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the cotreatment of BAY and TMZ significantly contributed to a decrease in the migration pattern of patientderived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatments outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the Stupp regimen radiation with daily TMZ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3 TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7 Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711 Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23 Both Bay and TMZ exert antitumoral activities individually in different tumor types28 Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31 to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35 Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCotreatment of Bay and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells ac LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells eg Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by cotreatment of Bay and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35 we further continued our experiments using patientderived GBM cellsApoptosis was promoted by cotreatment of Bay and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NFkB activity in LN229 U87 and patientderived GBM cell lines a NFkB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCotreatment of Bay with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54 To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60 However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnetts multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35 Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukeys post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo | 2 |
predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cellsmonocytes and macrophagesas they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung ï¬brosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389ï¬mmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid ï¬rstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a ï¬ner repertoireof self and nonselfantigens that facilitate pathogenspeciï¬c actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adeï¬ned enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have proammatoryproperties and androgens are thought to have antiammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory ï¬ow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that deï¬ne howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exempliï¬ed by tuberculosisor uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterones immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insuï¬cientcontrol of tissuedamaging ammatory responses seen inCOPD and pulmonary ï¬brosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantiï¬ed serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting ï¬nding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ¼ times those of DHEADHEA became of interest to the asthma ï¬eld because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled Eï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the ï¬rst second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary ï¬brosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both ï¬broblasts and alveolarepithelial cells Inï¬uenzaThe lungs are also the target of respiratory viruses such asuenza A ï¬u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to uenza A virus and to the uenzavaccine In general women have a more robust protectiveimmune response to uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identiï¬edthat high testosterone was correlated with a blunted responseto the ï¬u vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalï¬u is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how ï¬uctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune systemsresponse to the uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these ï¬ndings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during uenzainfection are lackingTuberculosisLike uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is deï¬nitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent speciï¬ctypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers oftheammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofproammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identiï¬ed as large highly autoï¬uorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary ï¬brosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to diï¬culties in their identiï¬cation and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of proammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentiï¬ed two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and deï¬ne thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to diï¬culties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited intoamed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and nonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into nonamed and amed tissues from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more eï¬cient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of tissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneï¬cialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16 are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identiï¬cation ofmonocytes in the lung compartments in humans has been achallenge because monocytic contamination from the bloodvessels Overcoming this challenge Desch et alperformed a ï¬ow cytometric phenotyping study and identiï¬edtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206 CD1c CD1a intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1c CD1a monocytes were described as tissue monocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erentammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erentammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a proammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as classical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or alternative activation of macrophages M2 macrophages produce ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models SimilarlyM2 macrophages are the predominant subset seen in pulmonaryï¬brosis and are responsible for ï¬brogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage ï¬uid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage ï¬uid of patients withCOPD Unlike in allergic asthma and pulmonary ï¬brosismacrophages in COPD are polarized toward an M1 proï¬le In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with deï¬ned cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldeï¬ned opposing phenotypes or does not ï¬tthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice and humans Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent proammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate | 2 |
expanding cancer predisposition genes with ultrarare cancerexclusive human variationsRoni Rasnic1 nathan Linial1 Michal Linial2It is estimated that up to of cancer incidents are attributed to inherited genetic alterations Despite extensive research there are still gaps in our understanding of genetic predisposition to cancer It was theorized that ultrarare variants partially account for the missing heritable component We harness the UK BioBank dataset of individuals of which were diagnosed with cancer to detect ultrarare possibly highpenetrance cancer predisposition variants We report on cancerexclusive ultrarare variations and nominate variants with additional independent evidence as cancer predisposition variants We conclude that population cohorts are valuable source for expanding the collection of novel cancer predisposition genesDiscovery of cancer predisposition genes CPGs has the potential to impact personalized diagnosis and advance genetic consulting Genetic analysis of family members with high occurrences of cancer has led to the identification of variants that increase the risk of developing cancer1 In addition to familybased studies efforts to identify CPGs focus on pediatric patients where the contribution of environmental factors is expected to be small Forty percent of pediatric cancer patients belong to families with a history of cancer2Tumorigenesis results from misregulation of a0one or more of the major cancer hallmarks3 Therefore it is anticipated that CPGs overlap with genes that are often mutated in cancerous tissues Indeed CPGs most prevalent in children TP53 APC BRCA2 NF1 PMS2 RB1 and RUNX12 are known cancer driver genes that function as tumor suppressors oncogenes or have a role in maintaining DNA stability4 Many of the predisposed cancer genes are associated with pathways of DNArepair and homologous recombination5 The inherited defects in cells ability to repair and cope with DNA damage are considered as major factors in predisposition to breast and colorectal cancers6Complementary approaches for seeking CPGs are largescale genomeexome wide association studies GWAS which are conducted solely based on statistical considerations without prior knowledge on cancer promoting genes7 Identifying CPGs from GWAS is a challenge for the following reasons limited contribution of genetic heritability in certain cancer types low effect sizerisk associated with each individual variant lowpenetrance in view of individuals background8 and low statistical power Large cohorts of breast cancer show that of cancer cases are associated with mutations in BRCA1 and BRCA2 which are also highrisk ovarian cancer susceptibility genes Additionally TP53 and PTEN are associated with earlyonset and highrisk familial breast cancer Mutations in ATM and HRAS1 mildly increase the risk for breast cancer but strongly increase the risk for other cancer types and a collection of DNA mismatch repair genes MLH1 MSH2 MSH6 PMS2 are associated with high risk of developing cancer9 A large cohort of Caucasian patients with pancreatic cancer reveal high risk CPGs that overlap with other cancer types CDKN2A TP53 MLH1 BRCA2 ATM and BRCA110Estimates for the heritable component of predisposition to cancer were extracted from GWAS familybased and twin studies11 These estimates vary greatly with maximal genetic contribution associated with thyroid and endocrine gland cancers and a minimal one with stomach cancer and leukemia14 Current estimates suggest that as many as of cancer incidents can be attributed to inherited genetic alterations eg single variants and structural variations1516 The actual contribution of CPGs varies according to gender age of onset cancer types and ethnicity17 It is evident that high risk variants with large effect sizes are very rare21 Actually based on the heritability as reflected in GWAS catalog it was estimated that only a fraction of existing CPGs is presently 1The Rachel and Selim Benin School of Computer Science and Engineering The Hebrew University of Jerusalem Jerusalem Israel 2Department of Biological Chemistry Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem Israel email ronirasnicmailhujiacilScientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 UK Biobank CUVs collection The Caucasian filtered UK Biobank UKBB data set include individuals who had cancer and the nonCaucasian include such individuals a Cancer type distribution for the Caucasian data set b Cancer type distribution for the nonCaucasian data set c The data of UKBB participants was used for this study of which were confirmed Caucasian d Out of UKBB variants we curated heterozygous and homozygous CUVs total CUVs known22 Therefore instances of extremely rare mutations with high risk for developing cancer remain to be discoveredA catalog of CPGs was compiled from a0years of research1 with about half of the reported genes derived from family studies representing highpenetrance variants An extended catalog was reported with a total of CPGs that were tested against rare variants from TCGA germline data covering cancer patients from cancer types and included known pediatric CPGs23 The contribution of BRCA12 ATM TP53 and PALB2 to cancer predisposition was confirmedIn this study we report on known and novel cancer predisposition candidate genes We benefit from the UKBiobank UKBB an invaluable resource of germline genotyping data for individuals The UKBB reports on cancer patients and cancer free individuals considered as control group We challenge the possibility that CPGs can be identified from very rare events henceforth called cancerexclusive ultrarare variants CUVs These CUVs are expected to exhibit high penetrance Notably the presented CUVs were extracted from UKBB DNA array and therefore only cover the array preselected SNPs We report on exome variations of which are heterologous The majority of the matching genes are novel CPG a0candidates We provide indirect genomic support for some of the CUVs that occur within coding genes and discuss their contribution to tumorigenesisResultsThe primary UKBB data set used in the is comprised of Caucasian UKBB participants see Methods Fig a01c cancerfree and diagnosed with at least one malignant neoplasm Among participants with cancer were diagnosed with either skin or breast cancer The clinical ICD10 codes assembly is summarized in Supplementary Table a0S1 A total of of the cancerdiagnosed individuals had two or more distinct neoplasms diagnosed The validation UKBB data set includes nonCaucasian participants among them are cancerfree Figure a01ab provide further details on different cancer type prevalence in these setsNonmelanoma skin cancer is mostly attributed to environmental factors rather than genetic association24 However based on evidence for hereditary links for nonmelanoma skin cancer predisposition2526 we included these individuals in our analysis In addition focusing on extremely rare variations enables the identification of existing yet overlooked genetic associationsCompilation of cancerexclusive ultrarare variants CUVs We scanned genetic markers in our prime data set for cancerexclusive variations variations met our initial criteria appearing at least twice in individuals diagnosed with cancer and not appearing in cancerfree individuals Among them were heterozygous and were homozygous variations In order to target variations with additional supporting eviScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Exomic CUVs are mostly gene disruptive The partition of variant types for the compiled list of exomic CUVs The list is dominated by transcript disruptive variations that include missense frameshift stop gain and splicing sites a Distribution of variation types among the exomic CUVs b Dispersion of variant types among heterozygous and homozygous CUVsdence we considered only coding exome and spliceregion variants To assure the CUVs rarity in the general population we applied an additional filter based on the gnomAD data set see Methods The resulting final list is comprised of variants associated with genes heterozygous and homozygous Fig a01d The detailed list of all CUVs can be found in Supplementary Table a0S2Most of the CUVs are missense variants There is a strong enrichment for loss of function LoF variants ie frameshift splicing disruption and stop gains which account for of the CUVs Only a single homozygous CUV is synonymous Fig a02a The distribution of variation types varies greatly between homozygous and heterozygous CUVs Fig a02b Missense variants are of the homozygous variant set but only of the heterozygous CUVs The heterozygous CUVs are highly enriched for LoF variants which constitute the other Cancerexclusive ultrarare variants overlap with known cancer predisposition genes From the listed CUVs variants were previously defined as cancer inducing genes in genes Table a0 Specifically CUVs within genes appear in the updated list of CPG catalog23 and CUVs within genes are known cancer driver genes Fig a03a as determined by either COSMIC27 or the consensus gene catalog of driver genes listing genes coined C29928 More than half of the cancer associated variants result in LoF Many of the affected genes are tumor suppressor genes TSGs among which are prominent TSGs such as APC BRCA1 and BRCA2 Table a0 each identified by two distinct CUVs Notably of the variants had at least one appearance in nonmelanoma skin cancerThe heterozygous CUVs are enriched for known cancer predisposition genes Twentyfive of the cancer associated CUVs are heterozygous and one is homozygous However there is an inherent imbalance in the initial variant sampling performed by the UKBB As the UKBB use DNA arrays for obtaining genomic data the identifiability of ultrarare exome variants is restricted by the selection of SNP markers and the design of the DNA array There are heterozygous ultrarare exome variants from genes which pass our biobankethnic and the gnomAD allele frequency filtration A total of of the filtered ultrarare variants overlap with known CPGs as some genes are overrepresented among the ultrarare variants Supplemental Table a0S3 For example the exomic region of BRCA2 is covered by such SNP marker variants while most genes have noneIn order to account for the disproportional number of the ultrarare variant of some CPGs we calculated the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the collection of heterozygous ultrarare variants As shown in Fig a03b there is an enrichment towards CPGs and even more so as we remove variants of overrepresented genes eg BRCA2 The statistical significance estimates pvalues for each datapoint are available in Supplemental Table a0S3 see MethodsIndependent genetic validation Due to the extremely rare nature of the CUVs we require additional support for the collection of the CPG candidates We seek independent genetic validation of the noncancer related CUVs We apply three sources for validation the filtered Caucasian UKBB cohort the matched filtered nonCaucasian UKBB cohort the collection of germline variants from TCGA as reported in gnomAD The complete list of genetically validated novel CPG candidates is listed in Table a0 Ten out of the novel CPGs were identified based on appearances in individuals with nonmelanoma skin cancerWithin the Caucasian cohort we consider the following as additional genomic evidence a gene with CUVs or any CUV seen in more than two individuals diagnosed with cancer We found genes that have distinct CUVs of which are already known CPGs BRCA1 BRCA2 and APC The other genes are likely novel Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cRefEffecthg19TMissenseGMissenseMissenseTSplice region GSplice region AFrameshiftFrameshiftStop gainMissenseFrameshiftMissenseStop gainMissenseMissense MissenseFrameshiftMissenseFrameshiftFrameshiftMissenseMissenseFrameshiftStop gainFrameshiftSplice region CMissenseTAlt GeneGBACMSH6AVHLGTGFBR2AMLH1GAPCAAGGA APCGTCTGTCC CTG AG TCTTCCGCACAGGCGAACAAGAGCTGGGCCACCGTCTGFBR1SPTAN1RETBMPR1APTENEXT2NUMA1ATMBRCA2BRCA2RB1ERCC5TSC2NF1BRCA1BRCA1TGIF1RUNX1NF2COSMIC C299 CPG FunctionaYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYEnzymeDNA repairUbqcomplexKinaseTSGTSGTSGKinaseCytoskeletalKinaseKinaseTSG PhosphataseTSG EnzymeMT Spindle poleDDR KinaseTSG DNA repairTSG DNA repairTSGDNA repairTSGRAS regulatorTSG DNA repairTSG DNA repairTGF ligandTFCytoskeletalYYYYYYYYYYYYYYYYYYYYTable CUVs overlap with known cancer predisposition or driver genes a Function abbreviation DDR DNA damage response TSG tumor suppressor gene TF transcription factor MT microtubule Ubq ubiquitin Variants with at least one appearance in nonmelanoma skin cancerFigure a0 CUVs list is enriched with cancer predisposition genes Out of the genes in the CUVs list are known cancer genes a Venn diagram of the genes associated with CUVs known cancer driver genes as reported in COSMIC and the consensus CPGs b Expected number of known CPG CUV orange versus the actual number of known CPG in heterozygote CUVs blue An unbalanced representation of genes in ultrarare variants of UKBB results in overrepresentation of some genes We therefore ranked the genes based on number of ultrarare variants Supplementary Table a0S3 For each rank we present the expected number of CUVs from CPGs and the actual number observed for CUVs from CPGsScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cGene SymbolZygote form People per CUV Distinct CUVs NonCaucasian cohortTCGA germlineAGR2AKR1C2DNAH3DSPEGFLAMENDOUHIST1H2BOHSPB2ICAM1ISLRKCNH2MAP3K15MRPL39MYBPC3MYO1ENAV3PCDHB16SARDHSCN5AWDFY4ZFC3H1HeteroHeteroHomoHeteroHeteroHomoHeteroHeteroHomoHomoHeteroHeteroHeteroBothHomoHeteroHomoHomoHeteroHeteroHomoYYYYYYYYYYYYYYYYYFunction in tumorigenesisAffects cell migration transformation and metastasis Wnt signaling tumor antigenExerts an inhibitory effect on oncogenesisCancer predisposed genes in Tunisian familyAffects cell adhesion Suppressed by TGFβPromotes matrix assemblyCancer biomarkerAffects major signaling pathwaysEpigenetically regulatedBiomarker under a clinical trialMarker for mesenchymal stem cells Deregulated gene in cancerAffects proliferation and migrationContributes to cell migrationTumor suppressor by targeting miR130Cytoskeletal modifierStimulates upregulation of motility and invasionActs as a suppressor of breast cancerActs as tumor suppressorPromotes breast cancer possess antipancreatic cancerPresentats viral tumor antigen on dendritic cellsIndirect activating DNA repairRefTable Novel validated CPG candidates Variants with at least one appearance in nonmelanoma skin cancerCPG candidates DSP KCNH2 MYBPC3 and SCN5A There are CUVs which we detected in three individuals with cancer Three of them are known predisposition or driver genes NF1 ATM and TGFBR2 The other genes are CPG candidates that were not previously assigned as such This set includes PCDHB16 DNAH3 ENDOU AGR2 HIST1H2BO and NAV3 Interestingly a certain homozygous CUV in the gene ICAM1 appeared in individuals with cancer in our filtered Caucasian cohortThe nonCaucasian UKBB cohort provides additional independent genomic evidence There are CUVs that appear at least once in an individual with cancer from the nonCaucasian cohort CUVs from the genes MYO1E SARDH and ISLR appeared in two distinct individuals with cancer from this nonCaucasian cohort while CUVs from PCDHB16 and known CPG BMPR1A appeared in a single individual with cancerTCGA germline variants were obtained using exome sequencing and thus offer an additional separate source for CUV validation Clearly the appearance of CUVs in TCGA germline data is not anticipated as we discuss variants that are ultrarare in both UKBB and gnomAD The TCGA collection within gnomAD includes only samples We identified CUVs that were also observed in TCGA gnomAD germline data one of a known cancer driver gene TGIF1 and novel CPG candidates PCDHB16 EGFLAM AKR1C2 MAP3K15 MRPL39 DNAH3 WDFY4 HSPB2 and ZFC3H1Based on the above support we compiled a list of validated CPGs which includes genes that are novel CPGs Among these genes CUVs are heterozygous are homozygous and MYBPC3 is supported by both heterozygous and homozygous CUVs Two of these genes have multiple validation evidence DNAH3 with a homozygous CUV which appears in individuals with cancer in the Caucasian cohort and within TCGA germline variant collection PCDHB16 with a homozygous CUV which appeared in individuals in the Caucasian cohort one individual in the nonCaucasian cohort and in the TCGA gnomAD resource In addition nonCPG cancerdriver genes with validated CUVs include TGFBR2 and TGIF1 that are also very likely CPG candidatesSome of the prominent genes in our list were signified by additional independent studies For example a novel oncolytic agent targeting ICAM1 against bladder cancer is now in phase of a clinical trial29 Additionally DNAH3 was identified as novel predisposition gene using exome sequencing in a Tunisian family with multiple nonBRCA breast cancer instances30Somatic mutations in novel CPGs significantly decrease survival rate There is substantial overlap between CPGs and known cancer driver genes Fig a03a This overlap suggests that somatic mutations in validated CPG candidates may have an impact on patients survival rate We tested this hypothesis for the novel CPG candidates Table a0 using a curated set of nonredundant TCGA studies compiled in cBioPortal3132 that cover patients By testing the impact of alteration in the novel CPGs in somatic data we expect to provide a functional link between the germline CPG findings and the matched mutated genes in somatic cancer samples Altogether of the patients had somatic mutations in one or more of the genes The median survival of patients with somatic mutations in these genes is a0months while the median for patients without Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Somatic mutations in CPG candidate effect cancer patient survival and disease progression The effect of somatic mutations in the novel CPG candidate Table a0 on the survival rate of TCGA cancer patients was tested via cBioPortal a MeierKaplan survival rate estimate b MeierKaplan diseaseprogressionfree estimatesomatic mutations in any of these genes is much longer a0months Applying the KaplanMeier survival estimate yields a p value of 178e in the Logrank test Fig a04a The KaplanMeier diseaseprogressionfree estimate was also worse for patients with somatic mutations in the novel CPGs with a p value of 603e Fig a04b Cancer types in this analysis are represented by varied number of patients and percentage of individuals with somatic mutations in any of the novel CPGs Supplemental Table a0S4 The trend in most cancer types match the presented pancancer analysis Survival and diseaseprogression estimate for each cancer type are available in Supplementary Figures a0S1S24 Hazard Ratios and confidence intervals were calculated see Materials and Methods and Supplemental Table a0S4We conclude that the CUVbased CPG candidate genes from UKBB carry a strong signature that is manifested in patients survival supporting the notion that these genes belong to an extended set of previously overlooked CPGsHomozygous variations are mainly recessive In order to ascertain whether the homozygous variations found are indicative of the heterozygous form of the variant as well we viewed the heterozygous prevalence within the UKBB Caucasian population In only a single variant in the gene MYO1E was the prevalence in healthy individuals significantly lower than in individuals with cancer p value As most of the variations have a strong cancer predisposition effect as homozygous variations it seems that their influence is explained by a recessive inheritance mode This phenomenon might explain the significant depletion of known CPGs within the homozygous variations in our listInspecting the heritability model of previously reported CPGs1 is in accord with our findings showing that while about twothirds of the genes comply with a dominant inheritance the rest are likely to be recessive Notably in the most updated CPG catalog of the genes were assigned with both inheritance patterns In our ultrarare list only MYBPC3 is associated with both heterozygous and homozygous variationsDiscussionWe present a list of CUVs from genes Among them variants from genes are associated with known cancer genes Most of these variants overlap with known cancer predisposition genes Expanding the number of currently identified CPGs is crucial for better understanding of tumorigenesis and identifying various processes causing high cancer penetrance Genetic consulting family planning and appropriate treatment is a direct outcome of an accurate and exhaustive list of CPGsKnown cancer predisposition variants only partially explain the cases of inherited cancer incidents CPGs identification has already impacted cancer diagnostics therapy and prognosis1 Genomic tests and gene panel for certain cancer predisposition markers are commonly used for early detection and in preventative medicine3334 It is likely that CPGs based on ultrarare variants are not saturated For example additional CPGs including CDKN2A and NF1 were associated with an increased risk for breast cancer35 Specifically CDKN2A has been also detected as a CPG in families of patients with pancreatic cancer36 Inspecting the function of genes associated with Scientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cthe identified genes further supports the importance of protein modification eg kinases and phosphatase function chromatin epigenetic signatures37 membrane signaling DNA repair systems and moreNumerous CUVs are present in individuals with nonmelanoma skin cancer For the most part nonmelanoma skin cancers are attributed to environmental factors Nevertheless studies show that there are in fact genetic components associated with the majority of nonmelanoma skin cancers2526 Accordingly CUVs can unveil such rare genetic associationsWe chose to focus on cancerexclusive variants to shed light on mostly overlooked ultrarare cancer predisposition variants Naturally additional ultrarare variants in the dataset are presumably cancer inducing Detecting these variants requires developing a broader model expanding the scope to somewhat less rare possibly lowerpenetrance variants The impending availability of UKBB exome sequencing exomes will enable us to revisit the identified variants to further refine the list of candidate CPGs ie removing falsepositives and adding evidence to support true CPGs and to develop a less strict detection modelThe inheritably rare nature of CUVs raise concerns on the reliability of their initial identification38 We overcome this hurdle by only considering as candidate CPGs those genes that are supported by additional independent genomic evidence from either the UKBB or the TCGA cohort We nominate genes as CPG candidates two of which are known cancer drivers As we have shown Fig a0 somatic mutations in the nondriver validated CPG candidates resulted in a significant negative effect on the patients survival rateMaterials and methodsStudy population The UKBB has recruited people from the general population of the UK using National Health Service patient registers with no exclusion criteria39 Participants were between and a0years of age at the time of recruitment between and To avoid biases due to familial relationships we removed samples keeping only one representative of each kinship group of related individuals We derived the kinship group from the familial information provided by the UKBB fam files Additionally samples had mismatching sex between the selfreported and the geneticsderived and samples had only partial genotypingWe divided the remaining participants into two groups Caucasiansindividuals that were both genetically verified as Caucasians and declared themselves as white nonCaucasiansindividuals not matching the previous criterion The Caucasian cohort includes individuals of whom had cancer and the nonCaucasian cohort includes individuals had cancer We used the Caucasian cohort for our primary analysis and the nonCaucasian cohort for additional validation purposesVariant filtration pipeline We considered a heterozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation and no healthy individuals with the variation in the Caucasian cohort We considered a homozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation ie homozygous to the alternative SNP and no healthy individuals with the homozygous variation in the Caucasian cohort The ensemble Variant effect predictor40 was used to annotate the variantsWe applied two additional filtration steps for the exomesplicingregion variants The first filter was applied using the nonCaucasian data set we filtered heterozygous variations with MAF and homozygous variations with homozygous frequency in this set This filtration step is meant to diminish variations which are mostly ethnic artifacts The second filter was applied to assure the variations rarity We applied the same filter heterozygous variations with MAF and homozygous variations with homozygous frequency using gnomAD v21141 The used gnomAD threshold was based on the summation of gnomAD v211 exomes and genomes We also used gnomAD for the TCGAgermline validation by extracting TCGA appearances from the databaseStatistical analysis The UKBB ultrarare variants are enriched with CPGs variants We accounted for this imbalance by calculating the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the ultrarare variant collection for heterozygotes We calculated pvalues for each datapoint using a twoside binomial testWe downloaded survival data from cBioPortal The data only included survival months We used Cox regression without covariates to calculate Hazard Ratio and confidence intervals The results are listed in Supplementary Table a0S4Rare variants reliability Our CUV collection includes variants that appeared at least twice in the filtered Caucasian cohort thereby evading many SNPgenotyping inaccuracies38 We further ascertain the validity of prominent variants with additional genomic evidenceCancer type definition The UKBB provides an ICD10 code for each diagnosed condition We considered an individual diagnosed with malignant neoplasm ICD10 codes C00C97 as individuals with cancer and otherwise as cancerfree individuals The codes were aggregated to improve data readability using the assembly described in Supplementary Table a0S1Ethical approval All methods were performed in accordance with the relevant guidelines and regulations UKBB approval was obtained as part of the project Ethical approval for this study was obtained from the Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0ccommittee for ethics in research involving human subjects for the faculty of medicine The Hebrew University Jerusalem Israel Approval Number UKBB received ethical approval from the NHS National Research Ethics Service North West 11NW0382 UKBB participants provided informed consent forms upon recruitmentData availabilityMost of the data that support the findings of this study are available from the UKBB However restrictions apply to the availability of these data which were used under license for the current study and so are not publicly available Data are available from the authors upon a justified request and with permission of the UKBB Data extracted from gnomAD is available from the authors upon requestReceived February Accepted July a1508 References Rahman N Realizing the promise of cancer predisposition genes Nature 101038natur e1298 Zhang J et al Germline mutations in predisposition genes in pediatric cancer N Engl J Med 101056NEJMo Hanahan D Weinberg R A Hallmarks of cancer the next generation Cell 101016jcell201102013 Vogelstein B Kinzler K W Cancer genes and the pathways they control Nat Med 101038nm108 Bertelsen B et al High frequency of pathogenic germline variants within homologous recombination repair in patients with advanced cancer npj Genom Med 101038s4152 Easton D F How many more breast cancer predisposition genes are there Breast Cancer Res 101186bcr6 Hindorff L A et al Potential etiologic and functional implications of genomewide association loci for human diseases and traits Proc Natl Acad Sci U S A 101073pnas09031 Galvan A Ioannidis J P A Dragani T A Beyond genomewide association studies genetic heterogeneity and individual predisposition to cancer Trends Genet 101016jtig200912008 Baria K Warren C Roberts S A West C M Scott D Chromosomal radiosensitivity as a marker of predisposition to common cancers Br J Cancer 101054bjoc20001701 Hu C et al Association between inherited germline mutations in cancer predisposition genes and risk of pancreatic cancer J Am Med Assoc 101001jama20186228 Verkasalo P K Kaprio J Koskenvuo M Pukkala E Genetic predisposition environment and cancer incidence a nationwide twin study in Finland Int J Cancer 101002SICI1097021519991 210836743AIDIJC830CO2Q Frank S A Genetic predisposition to cancerinsights from population genetics Nat Rev Genet 101038nrg14 Law P J et al Association analyses identify new risk loci for colorectal cancer susceptibility Nat Commun 101038s4146 w Czene K Lichtenstein P Hemminki K Environmental and heritable causes of cancer among million individuals in the Swedish FamilyCancer Database Int J Cancer 101002ijc10332 Economopoulou P Dimitriadis G Psyrri A Beyond BRCA new hereditary breast cancer susceptibility genes Cancer Treat Grant R C et al Prevalence of germline mutations in cancer predisposition genes in patients with pancreatic cancer GastroenRev 101016jctrv201410008 terology 101053jgastr o201411042 Petersen G M et al A genomewide association study identifies pancreatic cancer susceptibility loci on chromosomes 13q221 1q321 and 5p1533 Nat Genet 101038ng522 Wolpin B M et al Genomewide association study identifies multiple susceptibility loci for pancreatic cancer Nat Genet Long J et al Genomewide association study in East Asians identifies novel susceptibility loci for breast cancer PLoS Genet 101038ng3052 101371journ alpgen10025 Thomas G et al Multiple loci identified in a genomewide association study of prostate cancer Nat Genet 101038ng91 Mancuso N et al The contribution of rare variation to prostate cancer heritability Nat Genet 101038ng3446 Jiao S et al Estimating the heritability of colorectal cancer Hum Mol Genet 101093hmgddu08 Huang KL et al Pathogenic germline variants in adult cancers Cell 101016jcell201803039 Griffin L L Ali F R Lear J T Nonmelanoma skin cancer Clin Med J R Coll Physicians Lond 107861 Nikolaou V Stratigos A J Tsao H Hereditary nonmelanoma skin cancer Semin Cutan Med Surg 101016jclinm edici ne16162 sder201208005 Roberts M R Asgari M M Toland A E Genomewide association studies and polygenic risk scores for skin cancer clinically useful yet Br J Dermatol 101111bjd17917 Forbes S A et al COSMIC exploring the worlds knowledge of somatic mutations in human cancer Nucleic Acids Res 101093nargku10 cell201802060 Bailey M H et al Comprehensive characterization of cancer driver genes and mutations Cell 101016j Annels N E et al Phase I trial of an ICAM1targeted immunotherapeuticcoxsackievirus A21 CVA21 as an oncolytic agent against non muscleinvasive bladder cancer Clin Cancer Res 10115810780432CCR184022 Hamdi Y et al Family specific ge | 2 |
"The DICER algorithm (24) seeks one pair of linked modules at a time. A pair of modules is defined as linked if the sum of weights WG between them is high enough. We call the approach of DICER local as it finds one module pair at a time. The algorithm of Ulitsky et al. (17) aims to maximize the global score namely the total sum of scores within modules in H plus the sum of scores of links in G. In addition to increasing the global score links between modules are accepted only if they pass a statistical significance test. We call the second approach global. Both methods identify the links and the modules simultaneously.D demonstrates the differences between the local and global approaches. Assume that in both graphs edge weights are 1 non-edge weights are ?1 and that the local approach uses a threshold of 0 on the sum of WG weights between two modules for reporting a link. In both approaches modules are clusters of nodes with high density in H. According to both approaches module 1 is linked to module 2: the local score is 4 (8 edges and 4 non-edges) the global analysis P-value for linkage is <0.05 and the total score for the module pair is 13 (module score 6 + 3 + link score 4). The sum of WG weight between modules 2 and 3 is ?4 (10 edges and 14 non-edges) and the local method rejects that link. However the global approach will also link module 2 and 3: the linkage P-value is significant (P = 0.039) and adding this link will improve the global map score to 24 [13 for the (12) pair +15 for module 34 for the (23) link]. This example illustrates the advantage of the global approach on sparse graphs in which large modules are not expected to be densely interconnected.AlgorithmsWe conducted a systematic study and developed further a family of two-phase algorithms for module map detection that find an initial solution (possibly consisting of many small modules) and then improve it. We call algorithms for the first phase initiators and algorithms for the second phase improvers. For simplicity we describe the algorithms assuming that edges with positive weight are considered heavy. For unweighted graphs we assume edge weights to be 1 and non-edge weights to be ?1. For weighted graphs all node pairs (edges) have weights so there are no non-edges.InitiatorsWe tested five different initiators: (i) DICER (24) which finds one pair of linked modules at a time (ii) hierarchical clustering of the graph H (25) which finds a set of modules (iii) a greedy node addition algorithm for finding modules in H (iv) DICERk a variant of DICER wherein the minimum module size is set to k and (v) an algorithm based on enumeration of maximal bicliques in G using an exhaustive solver (2627) followed by the cleaning process of DICER. We call the latter algorithm MBC-DICER see Supplementary Text and Supplementary Figure S1 for a full description of all initiators. Each initiator creates an initial module set but modules in the map constructed by clustering algorithms are not necessarily linked.ImproversThe local improver (24) extends module map links by either adding a single node to a module or by merging two module map links. One drawback of this approach is that it cannot create new modules that are not represented in the initial solution. Another disadvantage is that it cannot merge a module whose two parts are linked to different modules that are unlinked. See Supplementary Figure S2 for examples." | 1 |
"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Marys Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturers recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patients space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patients blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patients space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to DisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturers recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturers instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c" | 2 |
LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR OBrien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree 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OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S 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thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL OHagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c' | 2 |
" laparoscopic tumorspecific mesorectal excision tsme preserving the left colic artery and superiorrectal artery is still a technically challenging procedure we conducted this study to demonstrate the feasibility ofthis procedure for upper rectal cancermethods a total of patients with upper rectal cancer were retrospectively analyzed in our cancer centerbetween april and april these patients were treated with either laparoscopic tsme n orlaparoscopic total mesorectal excision tme n in the tsme group the left colonic artery and superior rectalartery were preserved while they were not in the tme groupresults the operation time in the tsme group was longer than that in the tme group ± min vs ± min p furthermore the number of resected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p the blood loss between the tsme and tmegroups was not significant no mortality occurred in either the tsme or tme groups one patient in the tme groupunderwent conversion to laparotomy the total postoperative complication rates in the tsme and tme groups were and respectively there was no difference in severe complications between the two groupsanastomotic leakage and stenosiss laparoscopic tsme preserving the left colic artery and superior rectal artery can be safely conductedfor upper rectal cancerkeywords laparoscopic surgery rectal cancer tumorspecific mesorectal excision superior rectal artery leftcolonic artery tme correspondence 237721898qqcom 250537471qqcomhuxiang_zc1978sinacom chi zhang haotang wei wenqing hu and yueming sun contributedequally as joint first authors7department of general surgery yizhen peoples hospital clinical medicalcollege yangzhou university yangzhou jiangsu province china3department of gastrointestinal surgery changzhi peoples hospital theaffiliated hospital of changzhi medical college changzhi shanxi provincechina1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang world of surgical oncology page of introductiontotal mesorectal excision tme is an important surgical technique to prevent the local recurrence of rectal cancer on the other hand tme may not besuitable for every case of rectal cancer such as rectosigmoid junction and upper rectal cancers the resection range of tme reaches cm below the inferiorborder of the tumor and has acquired an adequatecure rate reported in previous studies for patientswith rectosigmoid junction and upper rectal cancers this tumorspecific resection according to thetumorsite or t staging is called tumorspecificmesorectal excision tsme itsudecks critical point at the rectosigmoid junction isdescribed as the point of origin of the last sigmoid arterial branch originating from the inferior mesenteric artery ima the anastomosis between the lastsigmoidal artery and superior rectal artery sra is absent in some people to avoid the risk of postoperativeischemic necrosis anastomotic leakage colitis and delayed stricturetosudecks point for cases where anastomosis may be absent or insufficiently present in addition the rate ofis desirable to ligate proximalabsence of the left colic artery lca is which maybe associated with a risk of anastomotic leakage due toinsufficient vascularization of the proximal colonic conduit this study introduces the procedure and technicalpoints of laparoscopic tsme with preservation of thelca and sra the operation is still a technically challenging procedure we conducted this study to demonstrate the feasibility of this procedure for upper rectalcancer and shortterm prognosismethodspatientslaparoscopic tsme preserving the lca and sra wasperformed on patients with upper rectal cancer fromapril to april in the same period patients with upper rectal cancer underwent standardtme surgery this study was conducted in accordancewith approved guidelines this study was approved bythe institutional review board of the first affiliatedhospital of dalian medical university written informedconsent was obtained from all patientsfig ima 3d cta 0czhang world of surgical oncology page of equipmentangled ° 10mm diameter 3d laparoscope insufflation equipment and bipolar electrosurgical deviceaesculap german harmonic vascular closure systemjohnson usa 10mm and 5mm port trocars teleflexmedical usa laparoscopic linear staplers mm inlength covidien usa hemolock polymer lockingsurgical clips teleflex medical usa and a circularstapler ethicon endosurgery usa were used in thisstudypreoperative preparationinferior mesenteric artery ima 3d cta examinationshould be performed before the operation to assess themesenteric vascular vessel types fig intestinal preparation was performed days before the operation andprophylactic intravenous antibiotics were used beforethe operation for min central venous catheterizationwas performed after general anesthesia the surgicalposture was the starboard lithotomy position with thehead lower and feet higherthe operating surgeon and camera assistant stood onthe patients right side and the first assistant stood atthe patients left foot side the laparoscopic monitor wasplaced on the patients right foot side the trocar for thelaparoscope was inserted from the right paraumbilicalside and four ports were used as working ports fig surgical techniquesthis surgical technique was characterized by thoroughlymph node dissection based on neurovascular preservation and dissection of the left colon and sigmoid andfig position of the trocarupper rectal vessels along the inferior mesenteric vesselsthe region of operation was the superficial layer of thenerve sheath on the vascular surface the left colonicand superior rectal vessels needed to be preserved andthe vascular branch from the sigmoid vessels and theblood vessels from the superior rectal vessels to the intestinal wall were selected and severed according to thetumor positionfirst we adopted a lateral approach by opening themonks white line along the descending and sigmoidcolon reaching the splenic flexure as the cephalad dissection point the correct plane of dissection wasachieved by toldts fascia we usually used bipolar electrosurgical devices and bipolar scissors to separate thiscorrect plane with gentle blunt and sharp dissectionthe ureter and other retroperitoneal structures weresafely protected by staying in this plane we continuedto dissect along the plane to the root of the ima thehypogastric nerves were visible the nerves were carefully protectedthen the dissection began at the position of the sacralpromontory the junction of the sigmoid mesentery andretroperitoneum from the previous dissection plane in thefirst step ideally we dissected the presacral space belowthe sra from the left side across the midline to the rightside attentively protecting the hypogastric nerves whileusing a bipolar electrosurgical device fig 3a the distaldissection endpoint was approximately cm below thetumor we needed to open the peritoneal reflection anddissect the lateral ligament of the rectum by protectingthe neurovascular bundle nvb using a harmonic vascular closure system in some patients we placed the dissected colon and mesocolon to the right celiac side andthoroughly revealed the left side of the mesocolon wecarefully employed dissection in the correct plane on thevessels to avoid tissue damage for the realization of enbloc resection the technique in this step is to identify therelationship between the left colic artery inferior mesenteric vein imv to the ima and sra and the branch ofthe arteriae sigmoideae fig 3b this vascular bundle canbe traced from the origin of the ima to the rectal segmentapproximately cm below the inferior border of thetumor fig 3cthe second step was performed using a medial approach this step involved thorough lymph node dissection based on neurovascular preservation the leftcolonic and superior rectal vessels need to be preservedand the sigmoid vessels and vessel branch from the superior rectal vessels to the intestinal wall were selectedand severed according to the tumor positiondissection at the correct presacral space and cephaladdissection to the ima could be employed our generalmedial approach was to begin at the presacral space andobtain a connection with the plane ofthe lateral 0czhang world of surgical oncology page of fig a dissection the presacral space below the superior rectal artery sra approached from the left side across the midline to the right sideattentively protected hypogastric nerves while using a bipolar electrosurgical device b identification of the relationship between left colic arteryimv to the ima and sra and the branch of the arteriae sigmoideae c tracing this vascular bundle from the origin of the ima to the rectumsegment approximately cm below the inferior border of the tumor d ligation of arteriae sigmoideae and vascular branch from sra eligation of arteriae sigmoideae and preserving left colonic vasculature f excision of the mesorectum just underneath the rectal wall about cm and avoiding injury to the rectal wall and sra g tsme preserving left colic artery and superior rectal arteryapproach pelvic dissection was performed from the entrance of the pelvic cavity down to the pelvic floor wecould identify both the hypogastric nerve fibers and pelvicnerve by using highdefinition 3d laparoscopy and preservethem the imvleft colic artery bundle was then carefullytraced to the junction position from the ima and lymphnode no253 was dissected the pelvic nerves and ureterwere already carefully insulated and the circumference ofthe ima could be revealed the mesocolon could be freedfrom the retroperitoneal position by anterior dissection bygently applying a bipolar electrosurgical device we dissected the sra and blood vessels from the sra to the intestinal wall and dissected lymph nodes no252 andno251 at this point we had completed lymph node dissection and completely clarified the relationship betweenthe lca imv ima sra and arteriae sigmoideae finallywe ligated the arteriae sigmoideae and vascular branchfrom the sra into the intestinal wall fig 3d while preserving the left colonic vasculature fig 3e energy devicesand hemolocks were used widely in this step 0czhang world of surgical oncology page of after the above procedure was completed we separated the rectal wall from the mesorectum with an adequate distance from the tumor in accordance with thet stage and position of the tumor using a harmonic vascular closure system in order to provide enough spaceto insert an endoscopic linear stapler we excised themesorectum about cm just underneath the rectalwall fig 3f careful surgery was performed to avoid injury to the rectal wall and sra then the endoscopic linear stapler was fixed the rectum was transected andsatisfactory tsme preservation of the left colic and superior rectal arteries was shown fig 3glastly a small 5cm incision was made at the leftlower abdomen and the specimen was taken outside ofthe abdomen and transected intraabdominal presacralanastomosis was performed by double stapling techniques after inserting the anvil head of a 28mm circularstapler into the oral side of the sigmoid colon doubledrains were placed and no diverting stoma wasperformedin the tme group the inferior mesenteric artery wassevered at the root the colon was severed cm awayand digestive tract reconstruction methods were similarto the tsme groupstatisticsspss190 version was used for statistical analysis categorical variables were compared using a Ï2 test continuous variables were presented as the mean standarddeviation or median range these variables were compared using a mannwhitney u test p values of were considered statistically significantresultsthe general characteristics of the included patients arelisted in table there were men and women in the tsme group and men and women in the tme group the meanage was ± years and ± years in thetsme and tme groups respectively there were no significant differences in preoperative comorbidity tumorsize depth of invasion and lymph node metastasis between groups the average distance between the tumorand anus of the tsme group was ± cm andthe distal margin was ± cm the pathologicalstages of the patients for the tsme group were as follows stage i stage iia stage iib stage iic stage iiia and stage iiib theproportion of patients with normal preoperative carcinoembryonic antigen cea was approximately of patients had cea levels between and ngml of patients had cea levels between and ngml and only patients had cea levels ngmltable clinicopathological features between the tsme andtme groupsfactorsage yearstme n ± tsme n ± p valuegendermalefemalebmi kgm2comorbidity ± ± cardiovascular disease respiratory diseasediabetes mellitushistological type differentiated typeundifferentiated type tumor size mm ± ± t categoryt1t2t3t4n categoryn0n1n2 conversion to open surgery operation time min ± ± blood loss ml ± ± lymph node dissection ± ± the operation time in the tsme group was longerthan that in the tme group ± vs ± p table furthermore the number ofresected lymph nodes in the tsme group was greaterthan that in the tme group ± vs ± p table the blood loss between groupswas not significantly different table the averagehospital stay in the tsme group was a little shorter thanthat in the tme group ± days vs ± days table no mortality occurred in either group one patient inthe tme group underwent conversion to laparotomy dueto bowel ischemia in the distal colon table the totalpostoperative complication rates in the tsme and tmegroups were and respectively table forsevere complications between the two groups anastomotic leakage and stenosis the severity of complicationswas claviendindo classification grades and therewas no significant difference between groups 0czhang world of surgical oncology page of tsme n tme n p value ± table postoperative complicationsfactorspostoperative hospital stay days ± mortalitymorbidityabsentpresentanastomotic leakagebleedingabdominal abscessileuswound infectionanastomotic stenosisurinary tract infectionascitesurinary retentionpneumoniacardiacrelated complications discussionin the british surgeon heald proposed tme for rectalcancer and pointed out that the anatomical level of tmewas clear so that the operative quality can be assessed the main concerns were a higher anastomotic leakage ratelonger operative time and higher blood loss after tme lopezkostner pointed out that tme was the standard operation performed for lower rectal cancers tme isnot necessary for cancers of the upper rectum therefore the tsme technique was introduced to achieve satisfactory local control and low morbidity partial mesorectalexcision is applied in tsme according to willians report in only of patients had distal intraluminal diffusion cm pollett and nicholls observed that there were no differencesin the local recurrence rate of rectal cancer between distal margins cm cm and cm a randomized prospective study of nsabb the national surgicaladjustburst and bowel project showed that the localrecurrence rate was not significantly different betweendistal rectal margins cm cm and cm according to the practice parameters for the management of rectal cancer edition a 2cm distal margin is more acceptable than cm but a 5cm distalmargin is still recommended total mesorectal resectiontme should be used for tumors located in the middleand lowerregardless oftwothirds ofwhether itis performed with low anterior resectionlar or combined abdominal and perineal resectionapr for tumors in the upper onethird of the rectumresection of the mesentery can be carried out accordingto the tumor situation and the distance between thethe rectumdistal margin and tumor should be cm the recommended grade was 1a tme was performed according to the distance between the distal margin of the rectal tumor and anus cm while tsme was performed for patients with adistance between the distal end of the rectal tumor andanus of cm in the authors medical departmentoncological outcomes after surgery can be divided intotwo aspects longterm survival and local recurrence ratelaw reviewed patients the 5year local recurrence rate for tme and partial mesorectal excisionpme for proximal cancer was and respectively the disease stage was associated with a higher riskof local recurrence there was no difference in the localrecurrence rates of tme and pme the 5year cancerspecific survival rates with and without tme were similarat and respectively kim reportedthat the 5year cancerspecific survival rate was andthe local recurrence rate was with cases of rectalcancer after tsme with pathologic stages iiii the riskfactors affecting cancerspecific survival rate were the ptstage pn stage positive distal resection margin and positive circumferential resection margin the risk factors affecting local recurrence were the pn stage positive distalresection margin and positive circumferential resectionmargin another study from a korean reviewed experience in patients with rectal cancer showed that theoverall local recurrence rate was the 5year local recurrence rates were and in stages i iiand iii respectively the 5year cancerspecific survivalrates were and in stages i ii and iiirespectively the risk factors were the pn stage and circumferential resection margin zakir performed an analysis with years of experience in rectal cancer patients who underwent laparoscopic andopen tsme surgery the 5year local recurrence rate was the overall 5year and cancerspecific survival rateswere and respectively there was no difference in the local recurrence rate between laparoscopic oropen resection the overall and cancerspecific survivalrates were and in the laparoscopic surgerygroup and and in the open surgery group respectively the results showed that laparoscopic surgerywas better than open surgery in overall and cancerspecific survival there was no difference in survival in patients with stage i however the survival rates in patientswith stages ii and iii among the laparoscopic surgerygroup were better than those in the open surgery groupwhich shows the superiority of laparoscopic tsme surgery for the longterm prognosis of rectal cancerkorean scholars conducted a study on the safety andprognosis of tsme after neoadjuvant chemotherapy forrectal cancer patients received 5fu with leucovorinchemotherapy and radiotherapy cgy for cycles 0czhang world of surgical oncology page of leadership was tsme was performed weeks later the resultsshowed that the overall complication rate was empiricalinternal construction was the 5year survival rate was and the 5yeardiseasefree survival was at present chinasouth korea and the usa have formulated similarguidelines for preoperative radiotherapy and chemotherapy for middle and low rectal cancer but there is nospecific reference data for preoperative radiotherapy andchemotherapy for upper rectal cancer the purpose ofthis paper is to introduce a new method of tsme anddiscuss the safety of the operation longterm survivaland local recurrence have not been discussedtsme surgery based on tme is now accepted as astandard for rectal cancer surgery and laparoscopic rectal cancer resection is accepted widely in the world eventhough it is a challenging procedure for surgery bloodloss in the laparoscopic group is well shown with anaverage of to ml the average blood loss inour study was ml lower than that reported in the literature we can identify neurovascular lesions usinghighdefinition 3d laparoscopy to preserve them and weuse a bipolar electrosurgical device to reduce injurywhich is beneficial for accurate operationthe overall complication rate in laparoscopic tsmeoperation was lower than that in the open operationgroup the rate of anastomotic leak showed no statistical difference between the two operation methods theaverage leak rate for rectal cancers was zakir reported that the overall complicationrate was in tsme for rectal cancer patients therate of anastomotic leakage was in the open tsmegroup and in the laparoscopic tsme group therewas no statistical difference between groups in our studythe incidence rate of postoperative anastomotic leakagewas three patients had complications after surgeryand the overall complication rate was the threecomplications were wound infection fluid collection andurinary retention with a claviendindo grading of yoo evaluated the optimal duration of urinarycatheterization after tsme for rectal cancer logistic regression analysis was performed to determine the risk factors for urinary retention the variables including age sexasa grade surgical procedure tnm stage tumor position preoperative radiotherapy duration of urinarycatheterization and time of surgery were not significantrisk factors for urinary retentionat present a 3d laparoscopic system aesculapgerman is used in laparoscopic surgery in our department single and reduced portlaparoscopic surgeryrobot operations and tatme operations are not usedfor tsme the surgeons who performed tsme had morethan years of experience in gastroenterostomy and hadexperience with open tsme the difficulty of the tsmeoperation is the management of the mesorectum seiji has reported on the management of the mesorectumin the narrow pelvis which our treatment method is basedon first the right part of the mesorectum is lifted fromthe right side of the sigmoid mesocolon to expose the inferior mesenteric artery and vein left colonic vessels sigmoid colonic vessels and superior rectum vessels theassistant lifts the left mesentery of the sigmoid colon exposes the above vessels expands the sigmoid mesocolonagain penetrates the mesentery from the right side andexposes the surrounding vessels expansion of the pelviccavity along the vessels is continued and the mesorectumis repaired from the left to the right side cm above thetumor according to the location ofthebranches of the severed vessels are determined and cm of the intestinal wall is repaired the rectum is dissected using an endogia staplerthe tumorlaparoscopic tsme has been used for rectal cancer andcan obtain satisfactory functional results compared to openresection and tme we do not think that the reduction inthe hospital stay is due to the acceleration of the intervention as per enhanced recovery after surgery eras butis due to an increase in the doctors confidence in reducingthe risk of postoperative complications after vascular preservation threedimensional cta examination is importantfor the preoperative evaluation of sigmoid colonvascular classification and intraoperative management ofthe sigmoid and left colon vessels however preoperativeexamination could not obtain information on the trafficbranch the biggest advantage of this operation is themaintenance of the blood supply of the proximal and distalintestines and the sufficient length of the intestine so thereis no need for temporary defunctioning stoma temporarydefunctioning stoma only increases the complexity of theoperation and closure of the temporary stoma increasesthe risk of complications in addition the results of the statistical analysis showed that the number of lymph nodes inthe tsme group was greater than that in the tme groupit cannot be concluded that tsme was significantly betterthan tme for lymph node dissection suggesting thattsme was not inferior to tmeslaparoscopic tsme with preserved left colic and superior rectal arteries is a technically challenging procedureintact visceral pelvic fibro is protected with even greateraccuracy than other techniques by 3d laparoscopywhich offers an optimal vision tsme with preserved leftcolic and superior rectum arteries did not increase therisk of operation compared with tme but increased thesurgeon s confidence in patient outcomes thereforelaparoscopic tsme with preserved left colic and superior rectal arteries can be safely performed for rectal cancer patients as an alternative to tme 0czhang world of surgical oncology page of abbreviationstme total mesorectal excision tsme tumorspecific mesorectal excisionima inferior mesenteric artery imv inferior mesenteric vein sra superiorrectal artery nvb neurovascular bundle pme partial mesorectal excisionacknowledgementsnoneauthors contributionslz yx and xh designed the study cz yx hw qz zd and whcollected and analyzed the data ma lz ys and xh interpreted the datalz and cz drafted the manuscript lz ma yx and xh revised themanuscript the authors read and approved the final manuscriptfundingthis study was supported by the jiangsu natural science foundationbk20180274 this funding supported the collection analysis andinterpretation of the dataavailability of data and materialsall experimental data used to support these findings are included in theethics approval and consent to participatethis study was approved by the institutional review board of the firstaffiliated hospital of dalian medical university written informed consent forpublication was obtained from all patientsconsent for publicationwritten informed consent was obtained from the patients and legalguardian for the publication of these patientscompeting intereststhe authors declare that they have no conflicts of interestauthor details1department of gastrointestinal surgery the first affiliated hospital of dalianmedical university dalian liaoning province china 2department ofgastrointestinal surgery the third affiliated hospital of guangxi medicaluniversity nanning guangxi province china 3department ofgastrointestinal surgery changzhi peoples hospital the affiliated hospital ofchangzhi medical college changzhi shanxi province china 4department ofcolorectal surgery the first affiliated hospital of nanjing medical universitynanjing china 5department of gastrointestinal surgery the first peopleshospital of dali city dali yunnan province china 6department ofgastrointestinal surgery graduate school of medicine university of tokyotokyo japan 7department of general surgery yizhen peoples hospitalclinical medical college yangzhou university yangzhou jiangsu provincechinareceived february accepted august heald rj husband em ryall rd the mesorectum in rectal cancer surgerythe clue to pelvic recurrence br j surg macfarlane jk ryall rd heald rj mesorectal excision for rectal cancerlancet lee ky factors influencing oncologic outcomes after tumorspecificmesorectal excision for rectal cancer j korean soc coloproctol williams ns dixon mf johnston d reapppraisal of the centimetre rule ofdistal excision for carcinoma of the rectum a study of distal intramuralspread and of patients survival br j durg pollett wg nicholls rj the relationship between the extent of distalclearance and survival and local recurrence rates after curative anteriorresection for carcinoma of the rectum ann surg wolmark n fisher b wieand hs the prognostic value of the modificationsof the dukes c class of colorectal cancer an analysis of the nsabp clinicaltrials ann surg monson jrt weiser mr buie wd chang gj rafferty jf prepared by thestandards practice task force of the american society of colon and rectalsurgeons practice parameters for the management of rectal cancerrevised dis colon rectum law wl chu kw anterior resection for rectal cancer with mesorectalexcision a prospective evaluation of patients ann surg kim sh bae kb kim jm oncologic outcomes and risk factors forrecurrence after tumorspecific mesorectal excision of rectal cancer cases j korean soc coloproctol kim nk min bs kim js hur h lee ky sohn sk oncologic outcomesand safety after tumorspecific mesorectal excision for resectable rectalcancer a single institutions experience with patients with rectalcancer j korean soc coloproctol zakir k mohamed wai lun law outcome of tumorspecific mesorectalexcision for rectal cancer the impact of laparoscopic resection world jsurg kim nk baik sh seong js oncologic outcomes after neoadjuvantchemoradiation followed by curative resection with tumorspecificmesorectal excision for fixed locally advanced rectal cancer impact ofpostirradiated pathologic down staging on local recurrence and survivalann surg poon jt law wl laparoscopic resection for rectal cancer a review annsurg oncol yoo be kye bh kim hj early removal of the urinary catheter aftertotal or tumorspecific mesorectal excision for rectal cancer is safe discolon rectum seiji o takashi t kazuki s a new laparoscopic surgical procedure toachieve sufficient mesorectal excision in upper rectal cancer int j surgoncol httpsdoi1011552011708439publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesenker w e thaler h t cranor m l polyak t total mesorectal excision inthe operative treatment of carcinoma of the rectum j am coll surg lopezkostner i lavery c hool gr rybicki la fazio vw total mesorectalexcision is not necessary for cancer of the upper rectum surgery zaheer s pemberton jh farouk r dozois rr wolff bg ilstrup d surgicaltreatment of adenocarcinoma of the rectum ann surg sudeck p ueber die gefässversung des mastdarmes in hinsicht auf dieoperative gangrän muenchen med wschr van tonder jj boon jm becker jhr anatomical considerations onsudecks critical point and its relevance to colorectal surgery clin anatcirocchi r randolph j cheruiyot i systematic review and metaanalysis of the anatomical variants of the left colic artery color dis httpsdoi101111codi14891 0c" | 0 |
" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [] an rln countof ¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1t3tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd students t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor cipreferenceage ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3rlnslymph node metastasislnr lodds ptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were à ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ¥ characteristicsunivariateormultivariateor cipreference age ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3lymph node metastasisptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ¥ deng proposed an optimal rln count of ¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncolamin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more personalized approach tocancer staging ca cancer j clin okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol tóth d bÃró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg 0c" | 0 |
among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patients health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patients quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal disquadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology 20e1 lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202adenocarcinomastech coloproctol 0c' | 0 |
" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncolli w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol lausen b schaumacher m maximally selected rank statistics biometricslausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p mantovani a allavena p sica a balkwill f cancerrelated inflammationnature giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncolli c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the | 0 |
"However the production and administration of these tailor-made DC vaccines are costly and labor-intensive [5]. As a next-step in the development of DC vaccines we designed a recombinant protein that contains a Mycobacterium tuberculosis heat shock protein 70 (MTBHsp70) fused to a single chain variable fragment (scFv) derived from human B cells that targets mesothelin. Mesothelin (MSLN) is a validated immunotherapy target that is highly overexpressed on the surface of common epithelial cancers including ovarian cancers epithelial malignant mesotheliomas ductal pancreatic adenocarcinomas and lung adenocarcinomas while expressed at relatively low levels only in mesothelial cells lining the pleura pericardium and peritoneum in healthy individuals [6-9]. Several therapeutic agents targeting MSLN are evaluated in preclinical and clinical studies such as the recombinant immunotoxin SS1P [9-11]. In our fusion protein the anti-MSLN scFv moiety was originally isolated from a yeast-display human scFv library [12] and demonstrated the ability to recognize both membrane-bound and soluble MSLNs and inhibit CA125/MSLN-dependent cell adhesion [13-15]. The recombinant MTBHsp70 protein provides immunostimulatory functions including the activation of monocytes and DCs to produce CC-chemokines that attract antigen processing and presenting DCs macrophages and effector T and B cells enhanced DC aggregation and maturation [1617] induction of the cytotoxic activity of natural killer cells [18] and improved cross-priming of T cells which is dependent on DCs [19]. The capabilities of MTBHsp70 as a potent immune adjuvant have been well characterized in cancer models including murine models of melanoma and lymphoma [1820-24]. While in these studies proteins or peptides fused with Hsp70 used for immunizations in mice were shown to generate humoral or cellular immune responses we expect that fusion of anti-MSLN scFv and MTBHsp70 takes advantage of the immune-activating action of MTBHsp70 and the tumor-targeting activity of the scFv which will yield anti-tumor responses against the broadest profile of tumor antigens. We evaluated the therapeutic efficacy of this MSLN-targeted fusion protein in syngeneic mouse models of ovarian cancer and mesothelioma and examined its mechanism of action in in vitro and in vivo cross-presentation assay systems. These studies demonstrate that this bifunctional fusion protein significantly enhances survival and slows tumor growth through the augmentation of tumor-specific cell-mediated immune responses. Results Expression of scFvMTBHsp70 fusion protein and MTBHsp70 The structure of scFvMTBHsp70 is shown in Figure 1A. VH and VL from anti-MSLN P4 scFv [13] are linked using a (G4S)3 linker and fused to full length MTBHsp70 with a (G4S)3 linker in between. As shown in Figure 1B only one protein band was observed with a molecular weight of approximately 100 kDa for scFvMTBHsp70 and one protein band with a molecular weight of 70 kDa for MTBHsp70 which match the expected molecular weights of these specific proteins. Endotoxin contamination levels in scFvMTBHsp70 and MTBHsp70 were found to be very low at less than 50 EU per mg of protein. Structure and analysis of scFvMTBHsp70 fusion protein. A anti-MSLN VH and VL are linked with a (G4S)3 linker and fused to full length MTBHsp70 with a (G4S)3 linker. B RAPIDstain based on Coomassie dye following purification and hIgG-Fc tag removal of MTBHsp70 and scFvMTBHsp70. C BR5FVB1 ovarian cancer cells and 40L mesothelioma cells were incubated with 40 ?g/ml scFvMTBHsp70 or 26 ?g/ml MTBHsp70 (blue line) or without either protein (solid) followed by anti-MTBHsp70 (IgG2a) biotinylated anti-IgG2a and Streptavidin-APC and then analyzed by flow cytometry. To confirm that the scFv portion of the fusion protein binds to MSLN on the surface of tumor cells scFvMTBHsp70 or MTBHsp70 was preincubated with 12 ?g/ml recombinant human MSLN for 30 min (red line) before being added to the cells. Data are representative of three independent experiments in duplicate tubes. D Median fluorescence intensity (MFI) values of cells stained with scFvMTBHsp70 or MTBHsp70 normalized to cells stained without either protein. Data are expressed as means?±?SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Turkeys multiple comparison tests. *p?<?0.05; **p?<?0.01;ns non-significant. E scFvMTBHsp70 binds with peritoneal mesothelial cells at a low level compared to ovarian cancer and mesothelioma cells. Binding of the fusion protein is at very low or undetectable levels on PBLs and splenocytes. Thick line with incubation of scFvMTBHsp70; solid without incubation of scFvMTBHsp70. Data are representative of three independent experiments. scFvMTBHsp70 binds to BR5FVB1 ovarian cancer cells and 40L mesothelioma cells through the interaction of scFv with MSLN on the surface of tumor cells Binding of scFvMTBHsp70 or MTBHsp70 to BR5FVB1 ovarian cancer cells or 40L mesothelioma cells as determined by flow cytometry is shown in Figure 1C and D. Binding of scFvMTBHsp70 to MSLN-expressing tumor cells was almost completely inhibited by preincubation of scFvMTBHsp70 with recombinant human MSLN. Although MTBHsp70 also binds to these MSLN-expressing tumor cells the level of binding is not significantly different from background (p?=?0.187 for BR5FVB1 cells and p?=?0.086 for 40L cells). Furthermore the binding of MTBHsp70 to cancer cells cannot be blocked by recombinant MSLN. These data support the view that binding of scFvMTBHsp70 to these tumor cells occurred via the interaction of the scFv portion of the fusion protein with MSLN on the surface of tumor cells. Binding of these proteins with 40L mesothelioma cells was further compared using fluorescence microscopy. scFvMTBHsp70 shows significantly stronger binding intensity as compared to MTBHsp70 (Additional file 1: Figure S1A and B). In order to determine if scFvMTBHsp70 also binds to normal tissue in addition to tumor cells we incubated the fusion protein with peripheral blood leukocytes (PBLs) splenocytes or peritoneal mesothelial cells from healthy FVB/NJ mice and stained the cells using the same method as was used for staining tumor cells. As shown in Figure 1E scFvMTBHsp70 binds with peritoneal mesothelial cells at a low level compared to ovarian cancer and mesothelioma cells. Binding of the fusion protein is at very low or undetectable levels on PBLs and splenocytes. Since scFvMTBHsp70 may potentially target peritoneal mesothelial cells we also explored whether it could induce inflammation in peritoneal mesothelial tissues. We injected na¯ve mice with saline scFvMTBHsp70 or MTBHsp70 plus P4 scFv at the same doses as those used for tumor therapy described in Method sacrificed the mice 7 days post final treatments and examined haematoxylin and eosin (H&E) stained sections prepared from abdominal and intestinal peritoneum. Light microscopic examination revealed no evidence of inflammation and no infiltration of inflammatory cells such as macrophages or granulocytic cells around the mesothelial cells lining the abdominal and intestinal peritoneum of the actively treated or control animals. Representative microscopic images are shown in Additional file 2: Figure S2. scFvMTBHsp70 significantly prolongs ascites-free survival and overall survival in ovarian cancer- or mesothelioma-bearing mice To determine whether scFvMTBHsp70 can prolong survival in tumor-bearing mice we first evaluated the protein in a syngeneic mouse model of papillary ovarian cancer using immune-competent FVB/NJ mice. As shown in Figure 2A scFvMTBHsp70 prolonged both ascites-free and overall survival time compared with saline or the equimolar mixture of MTBHsp70 plus P4 scFv. To further support the efficacy of this fusion protein in prolonging survival in MSLN-expressing tumor-bearing mice we evaluated this protein in a second syngeneic mouse model of mesothelioma using immune-competent C57BL/6 mice. Animals treated with scFvMTBHsp70 showed significantly prolonged ascites-free and overall survival time compared with saline- or MTBHsp70 plus P4 scFv- treated mice (Figure 2B). A and B Kaplan-Meier survival curves of tumor-bearing mice following treatment with scFvMTBHsp70 control proteins or normal saline. A In a syngeneic mouse model of papillary ovarian cancer in immune-competent FVB/NJ mice scFvMTBHsp70 prolonged ascites-free survival time compared with saline (n?=?10 per group representative of two independent experiments; median survival (Med. sur.)?=?47 days vs. 37.5 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 39 days). scFvMTBHsp70 also prolonged overall survival time in the mice compared with saline (Med. sur. = 51.5 days vs. 43 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 43 days). B In a syngeneic mouse model of mesothelioma in immune-competent C57BL/6 mice the fusion protein prolonged ascites-free survival time compared with saline-treated mice (n?=?20 per group pooled from two independent experiments; Med. sur. = 28 days vs. 26 days) or the mixture of MTBHsp70 plus P4 scFv (Med. sur. = 27 days). The fusion protein also prolonged overall survival time compared with saline (Med. sur. = 36 days vs. 31 days). P values were determined using the log-rank test. *p?<?0.05; **p?<?0.01; ***p?<?0.001. scFvMTBHsp70 enhances anti-tumor CD8+ T-cell responses in ovarian tumor-bearing mice To investigate whether the anti-tumor effects of scFvMTBHsp70 was associated with anti-tumor effector CD8+ T-cell responses we re-stimulated splenocytes from ovarian tumor-bearing FVB mice that received different treatments with the CD8+ T-cell Her2/neu epitope or MSLN Ld1 as a negative control ex vivo and analyzed the cells for production of IFN? and Granzyme B using flow cytometry. We previously showed that Her2/neu is expressed by BR5FVB1 cells [25]. Ld1 is an in-house designed H2d-restricted MSLN peptide that did not induce ovarian cancer specific T-cell response in H-2q FVB mice. We demonstrated significantly greater anti-Her2/neu CD8+ T-cell responses in splenocytes from scFvMTBHsp70-treated mice compared to mice treated with saline or a simple mixture of MTBHsp70 plus P4 scFv as measured by IFN? and Granzyme B production by CD8+ T cells (Figure 3A and B). This indicates that scFvMTBHsp70 enhances anti-tumor specific CD8+ T-cell responses in ovarian tumor-bearing mice. However no significant difference was seen in the number of tumor-infiltrating CD8+ T cells and no tumor-infiltrating Foxp3+ T cells were seen in tumors from mice in different treatment groups indicating that scFvMTBHsp70 may improve effector cell function rather than the number of intratumoral CD8+ T cells (Additional file 3: Figure S3A and B). Figure 3 Anti-tumor specific CD8+ T-cell functions in tumor-bearing mice following different treatments. A Splenocytes harvested from mice treated with scFvMTBHsp70 fusion protein equimolar mixture of MTBHsp70 plus P4 scFv or saline (n = 10 per group) were re-stimulated with Her2/neu peptide or MSLN Ld1 peptide. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Dunnetts multiple comparison tests. B Representative flow data are presented. C In vivo CD8+ T-cell depletion study. FVB/NJ mice were injected i.p. with anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a and were treated with scFvMTBHsp70 or saline as described in the methods. CD8+ T-cell depletion significantly and negatively impacted ascites-free survival in the scFvMTBHsp70 treated BR5FVB1 tumor-bearing animals compared to non depleted actively treated (n = 10 per group representative of two independent experiments; Med. sur. = 32.5 days vs. 48 days) animals. After CD8+ T cells depletion scFvMTBHsp70 treatment did not delay onset of disease (clinically evident ascites) compared with saline (Med. sur. = 32.5 days vs. 31.5 days; p = 0.5938). P values were determined using log-rank test. *p< 0.05; **p < 0.01 ***p < 0.001. scFvMTBHsp70 is able to prime an adaptive tumor-specific immune response that has an absolute requirement for tumor-specific CD8+ T cells To determine whether CD8+ T cells play a major role in the protective anti-tumor effects observed in mice treated with scFvMTBHsp70 we conducted in vivo CD8+ T-cell depletion experiments using monoclonal antibodies. The absence of circulating CD8+ cells in peripheral blood following depletion was confirmed by flow cytometry (Additional file 4: Figure S4A and B). As shown in Figure 3C CD8+ T-cell depletion significantly and negatively impacted ascites-free survival in the scFvMTBHsp70-treated BR5FVB1 tumor-bearing animals compared to non-depleted actively-treated animals. Following CD8+ T-cell depletion scFvMTBHsp70 treatment did not delay onset of disease (clinically evident ascites) compared to saline treatment. Therefore our data suggest that the priming of an adaptive tumor-specific immune response by scFvMTBHsp70 treatment is chiefly mediated by tumor-specific CD8+ T cells. scFvMTBHsp70 stimulates maturation of murine bone marrow-derived dendritic cells In order to investigate immunological mechanisms involved in the scFvMTBHsp70-enhanced anti-tumor immune response we first examined if the scFvMTBHsp70 or MTBHsp70 proteins used in our study could stimulate maturation of bone marrow-derived dendritic cells (BMDCs) as shown in previous studies [1617]. We stimulated CD11c+ BMDCs with 2 ?g/ml of scFvMTBHsp70 or an equimolar amount of MTBHsp70 (1.3 ?g/ml). 1 ?g/ml lipopolysaccharide (LPS) was used as positive control. To determine whether the BMDC maturation was attributable to LPS contamination of the recombinant proteins used in this study we also incubated BMDCs with 0.1 ng/ml LPS which was the equivalent amount of endotoxin found in 2 ?g/ml scFvMTBHsp70. After a 24 h-incubation both scFvMTBHsp70 and MTBHsp70 induced DC maturation indicated by an increase in the expression of CD40 CD80 CD86 and MHC class II molecules in comparison to the control cultures in medium. The increased expression of these DC maturation markers were comparable to those on cells stimulated with 1 ?g/ml LPS. The contamination control showed that addition of 0.1 ng/ml LPS did not replicate the effects of scFvMTBHsp70 or MTBHsp70 allowing us to discriminate the scFvMTBHsp70- or MTBHsp70-specific effects from effects of LPS (Figure 4A and B). Figure 4 scFvMTBHsp70 induces DC maturation and promotes antigen presentation and cross-presentation. A CD11c+ BMDCs isolated form FVB/NJ mice were incubated for 24 h with 2 ?g/ml scFvMTBHsp70 1.3 ?g/ml MTBHsp70 1 ?g/ml LPS as positive control or 0.1 ng/ml LPS as contamination control (thick lines) or medium only (solid) stained for CD11c CD40 CD80 CD86 and MHC II and analyzed by flow cytometry. Histograms were gated on CD11c+ DCs. Data are representative of three independent experiments in duplicate wells. B Median fluorescence intensity (MFI) of LPS- or protein-stimulated BMDCs normalized to MFI of BMDCs maintained in medium. Data are expressed as means?±?SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Dunnetts multiple comparison tests. C BMDCs cultured from FVB/NJ mice were pulsed with BR5FVB1 cells alone (Column a) or BR5FVB1 cells pre-complexed with MTBHsp70 (Column b) or scFvMTBHsp70 (Column c) and then incubated with BR5FVB1 tumor cell-primed T cells. Intracellular granzyme B and IFN? expressions in CD3+CD4+ and CD3+CD8+ T cells were analyzed by flow cytometry. Data from three independent experiments in duplicate wells are pooled and analyzed using One-Way ANOVA followed by Turkeys multiple comparison tests. Data are presented as mean?±?SEM. D Representative flow data are presented. E scFvMTBHsp70 enhanced tumor cell immunogenicity in vivo. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD4+ or CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Turkeys multiple comparison tests. *p?<?0.05; **p?<?0.01; ***p?<?0.001; ****p?<?0.0001. The scFvMTBHsp70 fusion protein increases tumor antigen presentation and cross-presentation by DC in vitro In the current study we demonstrated that splenic CD8+ T cells from scFvMTBHsp70-treated tumor-bearing mice could produce cytokines upon specific tumor antigen stimulation ex vivo which was associated with their antitumor therapeutic efficacy in vivo. To determine whether scFvMTBHsp70 promotes tumor specific T-cell responses by enhancing antigen presentation and cross-presentation by antigen presenting cells we co-cultured BR5FVB1 tumor cell-primed T cells with DCs that had been pulsed with BR5FVB1 tumor cells in the presence of scFv-MTBHsp70 MTBHsp70 or PBS. The scFvMTBHsp70/tumor cell-pulsed DCs induced significantly higher production of IFN-? and Granzyme B from both CD4+ and CD8+ tumor cell-primed T cells as compared with MTBHsp70 or PBS indicating that scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs (Figure 4C and D). scFvMTBHsp70 enhances tumor cell immunogenicity in vivo Having demonstrated in vitro that scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs we next explored whether scFvMTBHsp70 enhances tumor antigen presentation and cross-presentation by DCs and consequently enhances tumor cell immunogenicity in vivo. It has been demonstrated that the high density of DCs at dermal sites facilitates the capture of tumor antigens and that local inflammation induces DC maturation and migration into draining lymph nodes where they present antigens to na¯ve T cells generating a tumor specific immune response [26]. We primed FVB mice with an intradermal (i.d.) injection of mitomycin C-treated BR5FVB1 tumor cells followed by a booster i.d. injection of BR5FVB1 tumor cells with or without scFvMTBHsp70 or MTBhsp70. After 20 days we dissociated skin-draining lymph nodes and re-stimulated lymph node lymphocytes with Her2/neu peptides mitomycin C-treated BR5FVB1 tumor cells or BR5FVB1 tumor cell lysate and performed flow cytometric analysis for the presence of Granzyme B-generating CD4+ and CD8+ T cells. As shown in Figure 4E we demonstrated that Granzyme B-generating CD4+ and CD8+ T cells were significantly enhanced in mice that were immunized with scFv-MTBHsp70-bound tumor cells as compared to those in the mice immunized with tumor cells alone MTBHsp70-bound tumor cells or saline. Discussion We have developed a novel protein-based immunotherapy consisting of a fusion of an anti-MSLN scFv of human origin and recombinant mycobacterial heat shock protein 70 that has the ability to adjuvant significant T-cell responses against specific tumor antigens. P4 scFv directed against MSLN a surface antigen overexpressed on several types of tumor cells is used as a means of targeting the immunotherapeutic agent. We have demonstrated that this bifunctional fusion protein effectively binds BR5FVB1 ovarian cancer cells or 40L mesothelioma cells through the interaction of scFv with MSLN on the surface of tumor cells. We found that the fusion protein significantly prolonged survival time in syngeneic mouse models of papillary ovarian cancer and malignant mesothelioma. Treatment with the fusion protein induced significant tumor-specific CD8+ T-cell immune responses in the splenocytes of ovarian tumor-bearing mice. Furthermore in vivo CD8+ T-cell depletion studies demonstrated that this protective antitumor effect is mainly mediated by tumor-specific CD8+ T cells. Treatment using a mixture of MTBHsp70 plus P4 scFv for ovarian tumor or malignant mesothelioma-bearing mice did not increase survival or enhance tumor-specific immune responses suggesting that only through fusion of the two elements is the immune system effectively activated. We also demonstrated that this approach does not induce inflammation in the abdominal or intestinal mesothelial tissues as a result of a bystander interaction with MSLN on normal mesothelial cells. Several properties of MTBHsp70 appear in this study to contribute to the generation of tumor-specific CD4+ and CD8+ T-cell immune responses. First it induces maturation of DCs. Although several previous studies suggested that MTBHsp70 had pro-inflammatory properties only when contaminated with LPS [2728] other studies have decisively demonstrated that MTBHsp70 alone while not LPS promotes DC maturation and innate immune responses [161729]. In our study we used a fusion protein generated from a mammalian cell expression system ensuring a minimal amount of LPS contamination. We also incubated DCs with the same amount of LPS as that found in the fusion protein and failed to replicate the effects observed with the fusion protein supporting the view that maturation of DCs can be attributed to the fusion protein rather than LPS. Secondly MTBHsp70 is capable of delivering epitopes for enhanced processing and MHC-I presentation by DCs to na¯ve CD8+ T cells a process known as cross-presentation [30]. Mycobacterial Hsp70 fusion proteins have been shown to elicit both CD4+ and CD8+ T-cell responses although priming of CD8+ T cells does not appear to require CD4+ T cells [3132]. We demonstrated in this study that the MSLN-targeted fusion protein elicited significant tumor-specific CD8+ T-cell immune responses in ovarian cancer-bearing mice and this adaptive antitumor response has an absolute requirement for tumor-specific CD8+ T cells. Although at the dosing schedule used in these studies tumor-specific T-cell responses did not eventually lead to rejection of the established tumors they significantly prolonged survival time in tumor-bearing mice. DCs are believed to play a pivotal role in the initiation and programming of tumor-specific T-cell responses and are becoming an essential target in efforts to generate therapeutic immunity against cancer [33]. Two main approaches are currently under consideration for providing DCs with tumor-specific antigens. One approach is to culture patient-derived DCs ex vivo with an adjuvant that induces DC maturation in the presence of tumor specific antigens followed by adoptive transfer into the patient [33]. This approach is fraught with technical and practical difficulties such as selection of a suitable antigenic target inappropriate maturation state of selected DCs and the difficulty of generating a sufficient number of DCs ex vivo. In addition a number of investigators have recently reported that ex vivo-derived DC vaccines have an insignificant role in the direct priming of T cells in vivo[33-35]. An alternative approach to generate tumor-specific antigen bearing DCs is to induce them to take up tumor-specific antigens in vivo. It has been shown that in vivo specific targeting of tumor antigens to DCs improves the induction of antigen-specific CD4+ and CD8+ T-cell immunity. In these studies an agonistic anti-CD40 monoclonal antibody was used to mature DCs and eliminate antigen-specific tolerance [36-39]. MTBHsp70 has also been shown to stimulate inflammation and DC maturation via an interaction with CD40 receptors on both DCs and monocytes thus acting as an alternative ligand to CD40L [2940]. In our study we showed the fusion protein up-regulates surface expression of phenotypic markers of DC maturation. Interestingly in addition to CD80 CD86 and MHC class II molecules the expression of CD40 is also enhanced indicating a possible positive feedback loop involving CD40 signaling components. Beyond promoting DC maturation the scFvMTBHsp70 fusion protein also targets tumor cells towards the matured DCs. We propose that binding of the fusion protein with both tumor cells and DCs improves phagocytosis of parts of tumor cells by DCs and therefore any tumor antigen can be processed and loaded on both MHC class II and MHC class I molecules and presented to CD4+ and CD8+ T cells. This could explain the observed augmentation of tumor antigen presentation and cross-presentation brought about by the fusion protein in vitro. This may also explain the observed increased anti-Her2/neu CD8+ T-cell responses in the scFvMTBHsp70-treated ovarian tumor bearing mice although Her2/neu is not directly targeted. We recapitulated these in vitro findings in an in vivo tumor cell immunogenicity study. We used the fusion protein to activate and mature DCs in the skin such as Langerhans cells. These DCs then captured tumor cells or tumor cell fragments through the connection established by the fusion protein and migrated to the draining lymphoid organs where they presented tumor antigens to na¯ve T cells. T cells recovered from the draining lymph node showed significantly enhanced responses to stimulation with a range of tumor antigens. Conclusion Our study provides preclinical evidence that supports a protein-based immunotherapy that induces anti-tumor immune responses which normally require dendritic cell-based approaches. The MSLN-targeted MTBHsp70 fusion protein binds MSLN on tumor cells recruits and activates APCs including DCs loads DCs in vivo with the broadest profile of naturally processed tumor antigens promotes tumor antigen presentation and cross-presentation and enhances tumor specific CD4+ and CD8+ T-cell responses (Figure 5). Our study supports the continued exploration of this novel fusion protein alone or in combination with immune checkpoint inhibitors following conventional surgical reduction and chemotherapy for MSLN-expressing cancers. This new approach could significantly increase time to recurrence and survival in humans with ovarian cancer and mesothelioma where effective second line treatment options are very limited. Figure 5 A schematic model showing that the scFvMTBHsp70 fusion protein binds with MSLN on tumor cells and activates antigen presenting cells (APCs) thus promoting uptake of tumor cells or tumor cell fragments and promoting tumor antigen presentation and cross-presentation as well as adjuvanting tumor specific CD4 + and CD8 + T-cell responses. Methods Production of proteins The plasmid pQE30-MTBhsp70 that encodes full length MTBHsp70 was a generous gift from Dr. Peter Sveshnikov (Moscow Medical Academy Russia). The plasmid pTOR2-scFv that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein [13] generated and purified from yeast were generous gifts from Dr. Nathalie Scholler (Penn Ovarian Cancer Research Center University of Pennsylvania). The DNA fragment corresponding to a 15 amino acid linker (GGGGSGGGGSGGGGS) was connected to the scFv at its C-terminal using an overlap PCR approach. The PCR product scFv-linker was subcloned into pQE30-MTBhsp70 at the N-terminal of MTBhsp70. The DNA fragment for scFvMTBhsp70 was PCR amplified and cloned into pPMY5 (Promab) downstream of a human IgG1 Fc domain and separated from the Fc region by the signal cleavage sequence for Tobacco Etch Virus protease (TEV enzyme). scFvMTBHsp70 the MSLN-targeted fusion protein was generated from HEK293 cells and purified using Protein G resin (Pierce). The Fc region of the Protein G eluted protein was then cleaved from the fusion protein by TEV enzyme (Promab) digestion. MTBHsp70 was generated using the same expression system. The production and purification of these two proteins was accomplished by Promab Biotechnologies Inc. at Richmond CA. After purification and hIgG-Fc tag removal the integrity of scFv-MTBHsp70 and MTBHsp70 were determined by SDS-PAGE followed by staining with RAPIDstain (G-Bioscience). Endotoxin contamination levels in scFvMTBHsp70 and MTBHsp70 were determined by Limulus Amebocyte Lysate Assay (LAL-assay Cambrex). Cells The BR5FVB1 ovarian cancer cells a kind gift from Dr. Orsulic in Womens Cancer Research Institute at Cedars-Sinai Medical Center [41] or 40L mesothelioma cells a kind gift from Dr. Kane in Department of Pathology and Laboratory Medicine at Brown University [42] were maintained at 37°C in DMEM with 2 mmol/L L-glutamine 10 units/ml penicillin 10 ?g/ml streptomycin and 10% fetal bovine serum in humidified atmosphere with 5% CO2. Cells were cultured until 80% confluent and harvested with enzyme-free cell-dissociation buffer (Gibco) for in vitro tumor cell binding assays and cross-presentation studies or harvested with Trypsin EDTA (Mediatech) for animal injections. Mouse PBLs were obtained from FVB mice via tail vein bleeds after lysis of erythrocytes using M-lyse buffer (R&D systems). Small pieces of parietal peritoneal membrane were taken from the mice and digested in enzyme-free cell-dissociation buffer to obtain mouse peritoneal mesothelial cells. To test whether scFvMTBHsp70 or MTBHsp70 binds to the MSLN-expressing tumor cells or non-cancerous cells we incubated BR5FVB1 ovarian tumor cells 40L mesothelioma cells or normal cells from FVB mice including PBLs splenocytes and peritoneal mesothelial cells with 40 ?g/ml scFvMTBHsp70 or 26 ?g/ml MTBHsp70 followed by anti-MTBHsp70 (IgG2a) (Biodesign International) biotinylated anti-IgG2a (BD Bioscience) and Streptavidin-APC (BioLegend) and then analyzed the tumor cells by flow cytometry. As controls cells were incubated with the reagents described above except scFvMTBHsp70 or MTBHsp70. To confirm that scFv portion of the fusion protein binds to MSLN on the surface of tumor cells scFvMTBHsp70 or MTBHsp70 was preincubated with 12 ?g/ml of recombinant human MSLN (R&D Systems) for 30 min before adding to the cells. For fluorescence microscopy cells were cultured on coverslips until 50% confluent stained with 10 ?g/ml scFvMTBHsp70 or 6.5 ?g/ml MTBHsp70 followed by mouse anti-MTBHsp70 (1:500 dilution) and Donkey anti-mouse Alexa Fluor 594 (Invitrogen 1:500 dilution). Cells were observed using a Nikon Eclipse TiE fluorescence microscope. In some experiments tumor cells were treated with 20 ?g/ml mitomycin C at a concentration of 5 106/ml for 1 h in a 37°C water bath and washed with complete medium at least 3 times before use. Animal models and tumor treatment Ovarian cancer was established by i.p. injection of syngeneic cancer cells BR5FVB1 (107 cells per mouse) into 6-week-old female FVB/NJ mice as previously described [25]. All mice were purchased from Jackson laboratories. Intraperitoneal mesotheliomas were established by i.p. injection of syngeneic 40L cells (2??106 per mouse) into 6-week-old male C57BL/6 mice as previously described [42]. Mice with ovarian tumors were treated 7 days after BR5FVB1 tumor cell inoculation with i.p. injections of scFvMTBHsp70 (2 ?g per mouse) normal saline or an equimolar mixture of MTBHsp70 plus P4 scFv. This was followed by " | 1 |
"metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes Numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors Aspartate hydroxylase ASPH is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion ASPH also promotes tumor growth by stimulation ofangiogenesis and immunosuppression These effects are mainly achieved via the activation of Notch and SRCsignaling pathways ASPH expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact Therefore small molecule inhibitors of ASPH enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models ASPH can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation CD and CD4 T cells in mice The PAN3011 vaccine against ASPH has already been tested in aphase clinical trial in patients with prostate cancer In summary ASPH is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapyKeywords ASPH Small molecule inhibitor Metastasis ImmunotherapyBackgroundCancer is a multifactorial disease with an approximate million fatalities in Worldwide it is the secondleading cause of death [] The complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression Despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis [] delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer Since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential In thisapproachesthat Correspondence smahelmnaturcunicz1Department of Genetics and Microbiology Faculty of Science CharlesUniversity BIOCEV Vestec Czech RepublicFull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsAspartate hydroxylase ASPH has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] ASPH belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in of human solid tumors [] The overexpressed ASPH is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the Cterminal regionto the extracellular environment where it is accessible toantibody binding Recently molecular targeted therapyhas been developed against this target using small molecule inhibitors SMI that can inhibit the catalytic site The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKanwal Journal of Experimental Clinical Cancer Research Page of in the Cterminal region Moreover as antigenic epitopes that reside on the ASPH protein can efficientlystimulate cluster of differentiation CD and CD8 Tcell responses unique to tumor cells harboring ASPHthis enzyme can be used as a tumor associated antigenTAA in immunotherapy [ ]ThedioxygenasesStructure of the ASPH gene and isoformsASPH is a type II transmembrane protein of approxito the family of αmately kDa that belongsketoglutaratedependenthydroxylated products of ASPH hydroxylation were firstdetected in blood coagulation proteins [] ASPHwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbEGFlike domains of various proteins[] Fig Thereafter the human ASPH gene wascloned and characterized [] This gene spanning base pairs long region of genomic DNA and containing exons is located at the position q123 of thehuman chromosome The ASPH sequence is highlyconserved in mammalian evolution The sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species[] The whole ASPH protein consists of five domainsan Nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a Cterminal catalytic domain [] Tissue specific transcription is directed from two putative promoters P1 and P2which differ in their regulation sequences [ ] Whilethe transcription from the P1 promoter was observed inmost human tissues the P2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor MEF2 particularly in muscle tissues []The ASPH gene undergoes extensive alternative splicingresulting in four protein isoforms ie ASPH humbugjunctate and junctin [ ] These proteins vary in theCterminal region which affects their function [ ]The two longest ASPH transcript variantsthat aretranscribed from the P1 and P2 promoters and differ inthe length of the ²untranslated region encode the fulllength ASPH protein This protein contains the catalyticCterminal domain that catalyzes the posttranslationalhydroxylation in the cbEGFlike domains of numerousproteins Supplementary Fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] Thetruncated isoforms humbug junctate and junctin sharethe Nterminal part with the ASPH protein but lackcatalytic function They are involved in calcium homeostasis [] Humbug has a potential role in cell adhesionand calcium flux and similar to ASPH its overexpressionhas been correlated with aggressive tumorcell behavior[]reticulummembranebound protein that is known for its functionin the regulation of the intracellular Ca2 concentrationJunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicJunctateisaLocalization in cells tissue distribution andexpression regulationASPH is predominantly a cellsurface protein [] that isalso localized in the endoplasmic and sarcoplasmicreticulum [] Furthermore a recent study identifiedmitochondriallocalization of ASPH in hepatocellularcarcinoma HCC In that study ASPH overexpressioncorrelated with an instability of mitochondrial DNA andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in HCC []ASPH is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands [] and has a potential role in placentalimplantation and fetal growth [] On the contrary theASPH expression in normal adult tissues is relativelylow or negligible However ASPH expression is inappropriately activated during oncogenesis when ASPH is required for generation of malignant and metastaticphenotypes The elevated expression of ASPH at bothFig ASPH catalytic reaction Aspartyl and asparaginyl residues in cbEGFlike domains are hydroxylated 0cKanwal Journal of Experimental Clinical Cancer Research Page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer Table The first study that demonstrated thesignificantly higher expression of both ASPH mRNAand protein in HCC and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by Lavaissiere [] Subsequently they verified the role of upregulated ASPH protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the NIH3 T3 cell line and animal models []The level of ASPH also correlated with cell motility andinvasiveness in in vitro experiments [ ] In thestudy by Maeda [] the overexpression of theASPH protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients Similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung [] and colon carcinomas[] and glioblastoma multiforme [] Recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples ASPH has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes [] In glioblastoma the prognostic significance ofASPH was suggested by profiling of alternative mRNAsplicing []ASPH gene expression is upregulated via Wntcatenin [] and insulininsulinlike growth factor IGF1insulin receptor substrate IRS1 signaling [ ]through extracellular signalregulated kinaseERKmitogenactivated protein kinase MAPK andphosphatidylinositol3kinaseprotein kinase B PI3KAkt pathways Fig for review see ref [] InsulinIGF1IRS1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorsTable Summary of the studies which have identified the elevated ASPH expression in human tumor tissuesStudyPositive cases of studied samples nnTumor tissuesDetectionmethodIHCAntibody recognized region ofASPH proteinFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab NterminusFB50 Ab Nterminus or 15C7 Abcatalytic domainFB50 Ab NterminusmAb G3 hybridomaPolyclonalFB50 mAb NterminusIHCIHCIHCIHCRTqPCRIHCRTqPCRIHCIHCIHCRTqPCRIHCFB50 Ab NterminusLavaissiere []HepatocellularCholangiocarcinomaBreastColonPalumbo []Pancreatic adenocarcinomaSepe et al[]Primitive neuroectodermalmedulloblastoma neuroblastomaMaeda et al[]Cantarini []CholangiocarcinomaHepatocellularMonte et al[]HepatocellularYang et al[]Wang et al[]Dong et al[]Tang et al[]Lin et al[] types of tumor tissuesaHepatocellularPancreatic cancerHepatocellularBreast 75fold higher level of mRNAcompared to normal tissue 7fold higher level of mRNAcompared to normal tissuePancreatic ductal adenocarcinomaOgawa []aLiver kidney breast cervical ovarian Fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomaFB50 Ab NterminusIHC 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Regulation of ASPH expression and ASPH involvement in signaling pathways The expression of the ASPH protein can be regulated atseveral levels The ASPH gene can be amplified in tumor cells and its transcription activated by INIGF1 and Wnt catenin pathways or inducedby hypoxia At the posttranscriptional level miR200a and miR135a can downregulate ASPH expression Stability of the ASPH protein can bereduced by phosphorylation with GSK3 Conversely ASPH can enhance GSK3 activity by inhibition of its phosphorylation with AKT and p38kinases ASPH also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the Notch receptor and ligands ex JAG and interaction with pRb vimentin and ADAMs Finallyinactivation of NK cells by ASPH has been demonstrated Green arrow activation signal red bar inhibitory signal[] The catenindependent Wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies Upon aberrant activation of Wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus [] where an interaction between catenin and Tcellfactorlymphoidenhancerbinding factor TCFLEF proteins forms atranscriptional regulatory complex which enhances theexpression of Wnt target genes including IRS1 []ASPH was proposed as a common link between Wntcatenin and insulinIGF1IRS1 pathways and downstream signaling []The regulation of ASPH gene expression in tumorsmight also be affected by a copy number variation Inthe study by Kadota [] the ASPH gene locus hasbeen identified as one of the DNA regions with focalamplification in primary breast cancer In colorectal cancer ASPH gain or amplification was found in ofsamples [] Next a suppressant role of the microRNAmiR200a in posttranscription regulation of the ASPHexpression in hepatoma cells has been found [] MiR200a belongs to miR200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref[] Similarly miR135a has beenshown to suppress ASPH in endometrial cancer []Moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 GSK3casein kinase CK2 protein kinase A PKA and protein kinase C PKC on ASPH [] several studies demonstrated that phosphorylation can regulate the ASPHprotein expression [ ] Inhibition of the GSK3activity did not modify mRNA expression but increased 0cKanwal Journal of Experimental Clinical Cancer Research Page of the ASPH protein level [] Direct phosphorylation ofASPH by GSK3 probably decreases ASPH stability andthus reduces cell mobility [] ASPH protein expressionwas also increased by inhibitors of PKA PKC and CK2[] Mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofASPH phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] ThereforeASPH phosphorylation probably regulates the functionof this protein by various mechanismsASPH expression can also be regulated by hypoxia andoxidative stress In human neuronal cells this effect wasmediated by hypoxia inducible factor alpha HIF1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain PHD proteins and factorinhibiting HIF FIH are inactivated Consequently theHIF1 heterodimer made up of subunits HIF1α andHIF1 functions as a transcription factor likely enhancing ASPH expression by binding to hypoxiaresponsiveelements [] In hypoxic regions of glioblastoma bothHIF1α and ASPH were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of ASPH in mesenchymal transition [] Brewitz showed reducedASPH hydroxylation activity at low oxygen concentrations and suggested an ASPH role in oxygen hypoxiasensing ASPH upregulation induced by hypoxia couldcompensate for reduced enzymatic activity [] Moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon ASPHoverexpression which was reversed by silencing ASPHexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion []ASPH protein interactions and signaling pathwaysThe ASPH hydroxylation consensus sequence is confined within cbEGFlike domains that are found in proteins of diverse function including Notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3Axl family ofreceptor tyrosine kinases []The Notch signaling cascade is a remarkably conservedpathway Notch proteins Notch1 Notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment [] Notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells Dysregulation of the Notch pathway is directly linkedto cancer vascular disorders and congenital defects [] In mammals Notch signaling activated by binding ofone of two families of canonical Notch ligandsjaggedJAG1 and JAG2 and delta like DLL1 DLL3 and DLL4leads to the generation of the cleaved Notch intracellulardomain NICD fragment and its nuclear translocation Inthe nucleus the NICD fragment interacts with the DNAbinding complex CSL CBF1RBPjκ SuH Lag1 Thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bHLH transcription factor HES1 HESwith YRPW motif HEY1 CD44 epithelial cell adhesionmolecule EPCAM cmyc protooncogene matrix metallopeptidase MMP29 cyclin D1 cyclooxygenase vascular endothelial growth factor VEGF and proliferatingcell nuclear antigen PCNA [ ]Upregulation of ASPH results in enzymatic modification of the cbEGFlike repeats in the Notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof Notch signaling [ ] Furthermore the interactionof ASPH with a disintegrin and metallopeptidase domainADAM stabilizes this complex and enhances theS2 cleavage ofthe Notch receptors and subsequentNICD fragment release [] The activation of the targetgenes in malignant cells increases cell proliferation migration and invasion []through the epithelialtomesenchymal transition EMT that is probably upregulated by the interaction of ASPH with vimentin []Consequentlythis activation supports tumor growthand metastasis The ASPHNotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]The SRC kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis []ASPH has been identified as an SRC pathway activatorOverexpressed ASPH directly interacts with ADAM12 and strengthens the SRC activation by these proteinswhich promotes MMPmediated extracellular matrixdegradation and tumor invasiveness []ASPH can also contribute to malignant phenotype ofcells by interaction with other proteins Iwagami et alrevealed the interaction of ASPH with GSK3 that prevents GSK3 inactivation by phosphorylation with upstream kinases [] This mechanism was confirmed ina castrationresistant prostate cancer model [] GSK3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination Some types of tumors aresensitive to GSK3 inhibitors [] Recently Huang elucidated a direct binding of ASPH with retinoblastoma protein pRb leading to pRb phosphorylation[] They also showed that this effect was mediated byincreased binding of cyclindependent kinase CDK CDK4 and cyclins D1 and E with pRb and wasdependentAsASPH enzymaticonactivity 0cKanwal Journal of Experimental Clinical Cancer Research Page of phosphorylation of pRb inactivates its tumorsuppressorfunction ASPH can contribute to the progression of cellcycle via interaction with pRbEffect of ASPH on an immune systemTumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system [] In theseprocesses various mechanisms of both innate and adaptive immunity are included [] Immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties []As potential targets of ASPH hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when ASPH is overexpressed on cancercells Indeed such effect was demonstrated for humannatural killer NK cells by using recombinant ASPHwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively []Antibodies against ASPH inhibited these effectsInteraction of ASPH with other immune cells has notbeen studied However we suppose possible influence ofASPH on different tumorinfiltrating cells This assumption comes from the involvement of Notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells Forinstance Notch activation contributed to stimulation ofproinflammatoryantitumorigenic M1 polarization inboth bone marrowderived primary macrophages [] and tumorassociated macrophages[] WhenNotch signaling was abrogated protumorigenic M2polarization was induced even by stimulators of M1polarization [] miR125a has been identified as adownstream mediator of Notch signaling in macrophages [] Similarly the Notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of CD4 and CD8 T cells[] Different Notch receptors and their interactionwith different ligands contribute to these processes []Moreover noncanonical Notch signaling is implicatedin regulation of immune cells [] While activation ofNotch signaling in some cells eg T helper cells cytotoxic CD8 T cells and M1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory T cellsit leads to immunosuppression [] Thus immunostimulatory effect of Notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity [] Therapeutics affecting Notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedASPH as a therapeutic targetOncogenic abilities of ASPH have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with ASPHoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma [] pancreatic cancer [ ] glioma [] breast carcinoma [] castrationresistant prostatecancer [] and colorectal cancer [] In studies analyzingASPH function various approaches were utilized to revealsignaling pathways affected by ASPH Particularly ASPHexpression was diminished by using small interfering RNAs[ ] short hairpin RNAs [ ] or theCRISPRCas9 system [ ] The importance of ASPHenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by SMIs [ ] In vitro assays showed ASPH involvement in cell proliferation migration and invasion Cellularalterations included EMTinhibition of apoptosis andstemness acquisition Tumor growth and invasivenesscould further be supported by ASPHinduced extracellularmatrix degradation angiogenesis and transendothelial migration Notch and SRC signaling are probably major pathways influenced by ASPH Fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models Thus these studies also demonstrated thatASPH is a suitable target for cancer treatment especially bySMIs or immunotherapySmall molecule inhibitorsSMIs of ASPH Fig have been developed and used totest the role of ASPH in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] A small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches Not only can these inhibitors inhibit the catalytic activity of ASPH unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit ASPHcatalytic activity in the endoplasmic reticulum Differentcancers have different ASPH expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed []The first ASPH SMIs published were the tetronimidesMOI500 and MOI1100 Tetronimides were originallysynthesized in by Dahn [] and are redoxactivemimics of ascorbic acid and 2oxoglutarate MOI500 isa mixed inhibitor that inhibits both ASPH and the FatMass and Obesity protein FTO [] and is not onlyorally bioavailable but also can penetrate the blood 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig Small molecule inhibitors of ASPHthereand kinasesbrain barrier MOI1100 is a more potent inhibitor ofASPH and is also more selective [] Despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for MOI1100 Enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup [] as in MOI1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inMOI1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters MOI1182 isreported to have the ability to suppress invasive activityat a concentration of nM [] SMIs of ASPH have acharacteristic in vitro concentration dependent profilewhere the activity of the SMI plateaus at value around viability [] emphasizing the noncytotoxic properties of this class of inhibitorsNatural products and inhibitors of other enzymes thathave been repurposed as ASPH inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthineCN2019101414327 and guaianolides related to nortrilobolideCN2019104575886CN2019101414219CN2019101414187 0cKanwal Journal of Experimental Clinical Cancer Research Page of sesquiterpene with complex anticancerBosutinib is a wellknown inhibitor of BCRABL and SRCtyrosine kinases approved for the treatment of chronic myelogenous leukemia [] Cepharanthine is a natural productactivityincluding AMPactivated protein kinase AMPK activationand nuclear factor kappa B NFκB inhibition [] Nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium ATPase SERCA inhibition [] Recently a family of potent pyridine dicarboxylates have alsobeen published [] utilizing a mass spectrometrybasedinhibition assay [] These compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate The synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include PHD2 FIH and lysinespecific demethylase 4EKDM4E in addition to ASPH with varying degrees of selectivity However while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface ASPH inhibitors that may nothave cell penetrating activity []immunity As a target of humoralImmunotherapyASPH can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression Since ASPH is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityASPH on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity ADCC complement dependent cytotoxicity CDCor antibodydependent cellular phagocytosis ADCP []When the ASPH antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen HLA class Ior class II molecules and recognized by CD8 or CD4 Tlymphocytes respectively [] that can be stimulated byimmunization breaking tolerance to selfantigens []Induction of ASPHspecific CD4 and CD8 T cells wasexamined in blood samples of HCC patients Using synthetic peptides derived from ASPH after prediction of HLAclass I and HLA class IIrestricted epitopes it has beenfound that ASPH is a highly immunogenic protein that activates both types of analyzed T cells [] Thus efficientantitumor reactions could be stimulated by immunizationThe first vaccine against ASPH was based on matureddendritic cells DC loaded with the ASPH protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma [] This study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis BothCD8 and CD4 cells contributed to an antitumor effectinduced in a mouse model of HCC by immunizationwith ASPHloaded DCs []The next antiASPH vaccine was based on a bacteriophage lambda display system The viral capsid proteingpD was fused with the N or Cterminus of ASPH andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models []The vaccine PAN3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer[] This study demonstrated safety and immunogenicity of PAN3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen PSAor PSA doubling time ASPHspecific immune responseswere mediated by both antibodies and T lymphocytesAs ASPH is a type II transmembrane protein its Cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes Development ofASPHspecific antibodies has been described in several s [] The human IgG1 PAN622 recognizesthe catalytic domain of ASPH This antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells [] In the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results [] MouseIgG1 monoclonal antibody binding to the CterminalASPH domain mediated ADCC by human NK cells []Recently a secondgeneration antibody approach hasbeen disclosed The prepared antibody binds to the extreme Cterminus of ASPH US that is involved in specific substrate recognition [] Thereforethis antibody has direct ASPH inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activityPAN622 wasbioimagingusedforConclusionsASPH is an important enzyme in malignant transformationof cells It stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment Two main pathways Notch and SRCthrough which ASPH promotes the tumor growth havebeen identified It has also been shown that ASPH expression is induced by some growth factors and hypoxia and isregulated at various levels The overexpression of ASPHand its downstream targets has been detected in numeroushuman malignancies Since ASPH is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsFig Small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0cKanwal Journal of Experimental Clinical Cancer Research Page of Fig ASPH as a therapeutic target ASPH expression is upregulated by growth factors and hypoxia Its enzymatic activity can be inhibited bySMIs or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionConsequently tumor growth and metastasis are also reduce | 2 |
complex disease caused by coordinated alterations of multiple signaling pathways TheRasRAFMEKERK MAPK signaling is one of the bestdefined pathways in cancer biology and its hyperactivation isresponsible for over human cancer cases To drive carcinogenesis this signaling promotes cellular overgrowthby turning on proliferative genes and simultaneously enables cells to overcome metabolic stress by inhibitingAMPK signaling a key singular node of cellular metabolism Recent studies have shown that AMPK signaling canalso reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components RAFKSRfamily kinases which affects not only carcinogenesis but also the outcomes of targeted cancer therapies againstthe MAPK signaling In this review we will summarize the current proceedings of how MAPKAMPK signalingsinterplay with each other in cancer biology as well as its implications in clinic cancer treatment with MAPKinhibition and AMPK modulators and discuss the exploitation of combinatory therapies targeting both MAPK andAMPK as a novel therapeutic interventionKeywords RasRAFMEKERK signaling AMPK signaling Interplay Tumorigenesis Cellular metabolism RAFMEKERKinhibitors AMPK inhibitors AMPK activators Autophagy Targeted therapyIntroductionThe RasRAFMEKERK MAPK signaling is a fundamental pathway in cell biology and its alteration causeshuman cancers or developmental disorders Given its crucial roles in physiology and pathology this pathway hasbeen extensively studied for over two decades Unfortunately the regulation of MAPK signaling remains ambiguous till now by virtue of its intrinsic complexity anddiverse crosstalks with other signalings Here we focus onthe complicated interplays between the MAPK and theAMPK signalings in cellular carcinogenesis and their implications in current targeted cancer therapies We hopethis review would provide a conceptual framework for Correspondence yuanjiminszhospitalcom hujianchengnccscomsg1Department of Urology Shenzhen Peoples Hospital The Second ClinicalMedical College Jinan University The First Affiliated Hospital SouthernUniversity of Science and Technology Shenzhen Guangdong China4Cancer and Stem Cell Program DukeNUS Medical School College RoadSingapore SingaporeFull list of author information is available at the end of the developing more effective therapeutic approaches againsthyperactive MAPK signalingdriven cancersThe RasRAFMEKERK MAPK signaling and itsaberrant activation in cancersThe RasRAFMEKERK MAPK signalingThe RasRAFMEKERK MAPK mitogenactivated protein kinase signaling is a central pathway that regulatescellular proliferation differentiation and survival Thissignaling pathway was discovered in the 1970s1980swhen Ras small GTPases were identified as first oncogenes from sarcoma viruses [] Later studies on viraloncogenes had also led to the discovery of a Nterminaltruncated version of RAF SerThr kinase RAF1 or CRAF[] In contrast the other two components of this signaling pathway MEK mitogenactivated protein kinasekinase and ERK mitogenactivated protein kinase wereidentified as cytoplasmic protein kinases activated by mitogens in the 1990s [] Following these discoveries The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYuan Journal of Hematology Oncology Page of RAF was identified as the upstream kinase of MEK in and the first direct effector of Ras in [ ]resulting in the delineation of the whole MAPK signalingpathway which is considered as a milestone in our understanding of how cell senses external stimuliThe first component of MAPK signaling Ras smallGTPases have three gene isoforms Hras Kras and Nras that encode four proteins with splicing isoforms ofKras giving rise to Kras4A and Kras4B Although allRas proteins possess highly homologous sequences theyhave quite different activities tissue expression patternsand effector preferences which lead to their differentialphysiological and pathological functions []The downstream of Ras small GTPases is the RAFMEKERK kinase cascade [] The first kinases in thiscascade RAFKSR kinase suppressor of Ras family kinases include three RAF isoforms ie CRAF BRAF andARAF and two close pseudokinases ie KSR1 and KSR2All RAF isoforms have highly homologous sequences andsimilar structures with three conserved regions conservedregion CR1 contains RASbinding domain RBD anda Cysrich domain [ ] conserved region CR2 ischaracterized by a SerThrrich sequence conserved region CR3 comprises of a putative kinase domain with aNterminal acidic motif NTA [] and a Cterminalregulatory tail [] Nevertheless RAF isoforms havevariable kinase activities with an order as BRAFCRAFARAF likely by virtue of their distinct NTA motifs andAPE motifs that contribute to the dimerizationdriventransactivation of RAFs [] In contrast to RAF isoforms KSR proteins replace the RBD at the Nterminusfused sterile αmotif and Prorichwith a coiledcoilstretch that are responsible for recruiting proteins to theplasma membrane upon stimulation and lack the catalyticlysine in VAIK motif of kinase domain which impairs theircatalytic activity [ ] Given their associations withMEK and ERK as well as low kinase activity KSR proteinshave been thought as scaffold proteins in a long termHowever recent studies have indicated that KSR proteinscan also function as allosteric activators to stimulate thecatalytic activity of RAF proteins through dimerization[ ] The sidetoside dimerization of RAFKSRfamily kinases is critical not only for their activation butalso for their catalytic activity towards downstream kinases [ ] MEKs MEK1 and MEK2 are the second kinases of the RAFMEKERK kinase cascade whichhave both redundant and nonredundant functions [] These two dualspecific kinases comprise a shortregulatory Nterminus and a canonic kinase domain TheNterminal regulatory region of MEK12 contains a docking site for substrate ERKs a nuclear export sequence thatcontrols the cytoplasmicnuclear shuttling of proteins anda negative regulatory sequence that forms a helix andlocks kinase in an inactive conformation [ ]Further through its kinase domain MEK12 forms a facetoface heterodimer with RAFKSR or a homodimerheterodimer with itself which is indispensable for its activation stimulated by RAF and for its activity towards ERKs[ ] Like MEKs the terminal kinases of MAPKsignaling ERKs also include two highly homologousmembers ERK1 and ERK2 which have a central kinasedomain flanked by short N and Cterminal tails Thesetwo isoforms also have redundant functions albeit different expression patterns [] However unlike RAFs andMEKs that have very limited substrates ERKs recognizeand phosphorylate numerous substrates that include transcription factors protein kinases and phosphatases andother functional proteins []It should be noted that active Ras also turns on othersignaling pathways such as PI3KAKTmTORC whichregulate different cellular functions [] In this reviewwe focus only on the MAPK signaling given its dominant role in cancer biologyHyperactive RasRAFMEKERK MAPK signaling incancersThe MAPK signaling plays a crucial role in cell biologyand is tightly regulated in normal cells Upon engagement of receptor tyrosine kinases RTKs or other stimulations Ras small GTPases are activated by GTPGDPexchange factors GEFs which in turn recruit RAFMEK complexes to the plasma membrane and triggerthe RAFMEKERK kinase cascade through facilitatingRAFRAF or KSR RAFMEK and MEKMEK interactions as well as subsequent phosphorylations [] ActiveERKs are further translocated into the nuclei or stay inthe cytoplasm where they phosphorylate a number ofsubstrates that regulate cell functions [ ]On the other hand active MAPK signaling also turns onsome negative feedback loops which help cells return toquiescent status [] An aberrant activation ofMAPK signaling frequently induces human cancers ordevelopmental disordersthough an extremely highMAPK signaling may induce cell death or senescenceunder some conditions []its upstream activators or componentsHyperactive MAPK signaling exists in over ofcancers which is caused directly by genetic alterationsofincludingRTKs Ras and BRAF or indirectly by those independent of Ras or RAF [] and significantly promotesdisease progression [] Since genetic alterations ofRTKs in cancers have been extensively reviewed in recent years [] here we focus on oncogenic mutations of Ras and BRAF As a small GTPase Ras cyclesbetween active GTPbound status and inactive GDPbound status which is regulated by GEFs and GTPaseactivating proteins GAPs Oncogenic Ras mutationscan be mainly classified into two groups mutations 0cYuan Journal of Hematology Oncology Page of on glycine or G1213 that impair GAP associations and mutations on glutamine Q61 that diminish the intrinsic GTPase activity of Ras [] both ofwhich lead to an extended halflife of GTPloaded RasOncogenic Ras mutations have both isoform andcancertype preferences Kras is mostly mutated in allcancers followed by Nras and Hras and its mutations prevailin pancreatic cancers whilethose of Nras in myeloma and melanomas and Hrasin adrenal gland cancers [ ] This phenomenon mayreflect underlying fundamental signaling landscapes andRAS mutants interplay with these landscapes As thedownstream effector of Ras RAF is another dominanttarget of oncogenic mutations in the MAPK signalingpathway Similarly RAF mutations have isoform preference in cancers as Ras mutations with BRAF CRAF ARAF which may arise from their different basal activities Overall a single point mutation that converts Val into Glu in the activation loop of BRAF accounts for cases [] Although BRAF V600E exists only in of all cancers it is highly prevalent in some tissuespecific cancers such as melanoma thyroid cancer and histiocytosis [] albeit theunderlying molecular mechanisms remains unknown Incontrast to Ras and RAF MEK and ERK have rare mutations in cancers though their mutations have been shownto be responsible for some RAF inhibitor RAFiresistantcases in current cancer therapies []Targeting the RasRAFMEKERK MAPK signalingpathway for cancer therapy promising but challengingGiven their high prevalence in cancers great efforts havebeen made to develop specific inhibitors against oncogenicRas and RAF mutants in the last decades These inhibitorsthat have been approved for clinic treatment of RasRAFmutated cancers or under clinical trials are listed in Table However none of these inhibitors can effectively target thelarge portion of Ras mutants in cancers Since having no attractive docking sites suitable for designing highaffinity andselective small molecule inhibitors Ras mutants have beenthought as undruggable cancer drivers in a long term Untilrecently a group of covalent small inhibitors that are dockedinto a previously unknown pocket of GDPbound Ras andare linked to the adventive cysteine of RasG12C have beendeveloped and achieved encouraging outcomes for treatingRasG12Cdriven cancers as a single agent in clinical trials[] Fig To further enhance their efficacy theseRasG12C inhibitors are also undergoing clinical evaluationwhen combined with SHP2 Src homology region domaincontaining phosphatase2 inhibitors that block the pathwayreactivation caused by the relief of negative feedback loops[ ] Clinical Trial NCT04330664 In addition these inhibitors have also been further developed into RasG12C degraders by conjugating with ligands of ubiquitin E3 ligaseswhich effectively deplete Ras mutant proteins in cancer cells[ ] though their efficacy in vivo remains unknown Unlike RasG12C the majority of Ras mutants remain undruggable at present []It has been shown that Ras activates downstream effectors through direct interactions Therefore disruptingRaseffector interactions might be an alternative approach that can effectively block cancer growth drivenby Ras mutations Such a type of small moleculeblockers include rigosertib sulindac and MCP110 andamong which the therapeutic efficacy of rigosertib combined with nivolumab for Rasmutated cancers is beingdetermined by phase III clinical trials currently []Clinical Trial NCT04263090 Howeverit has to benoted that these inhibitors impair the MAPK signalingin both Rasmutated cancers and normal tissues andthereby their therapeutic index may not be highGenetic studies have revealed that the ablation of theRAFMEKERK kinase cascade but not other effectorpathways is a most efficient approach to inhibit thegrowth of Rasmutated cancers [] which leads to extensive developments of specific inhibitors against thiskinase cascade for treating Rasmutated cancers Moreover these inhibitors should be also effective for treatingRAFmutated cancers Indeed a number of RAFMEKERK inhibitors have been developed and applied to clinical trials for treating RasRAFmutated cancers [ ] At present three RAF inhibitors and three MEKinhibitors have been approved to treatlatestageBRAFV600Eharboring cancers as a single agent or incombination with other chemotherapeutics and exhibited excellent efficacies [ ] Fig HoweverRasmutated cancers possess intrinsic resistance to bothRAF and MEK inhibitors [] and even BRAF V600Eharboring cancers develop acquired resistance after months treatment [ ] Mechanistic studies haveshown that active Ras facilitates the RAF dimerizationon plasma membrane which leads to both intrinsic andacquired resistance to RAF inhibitors [] Toovercome the drug resistance arising from enhancedRAF dimerization the secondgeneration RAF inhibitorssuch as PLX8394 BGB283 TAK580 and CCT3833have been developed and are undergoing clinical evaluations Clinical Trials NCT02428712 NCT02610361NCT03905148 NCT02327169 NCT02437227 Thesenovel RAF inhibitors reduce the RAF dimerizationdriven resistance through distinct mechanismsPLX8394 and BGB283 impair RAF dimerization uponloading on RAF proteins [] TAK580 bindsto and inhibits both protomers in RAF dimers [] CCT3833 inhibits both RAF and upstream kinases ofRas and thereby prevents the activation of Ras by the relief of negative feedback loops [ ] Besides thesesecondgeneration RAF inhibitors a unique RAFMEK 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancersTargetKRasG12CDescriptionPhase I results showed ORR of nonsmall cell lung cancer NSCLC harboring KRas G12CCompoundAMG510Development stagesPhase IIINCT04303780MRTX849JNJRigosertibRasPhase IIINCT03785249Phase IIINCT04330664Evaluation of clinical activity of MRTX849 alone and combined with TNO155SHP2 inhibitor in KRas G12C mutated cancersPhase I NCT04006301 Safety and PK of JNJ74699157Phase IIINCT04263090Evaluation of safety and clinical efficacy of Rigosertib plus Nivolumab PD1 Abin KRas mutated NSCLCBRAFVemurafenib ApprovedLatestage or unresectable melanoma expressing BRAF V600E in ErdheimChester disease ECD with BRAF V600E mutation in DabrafenibApprovedEncorafenibApprovedLatestage or unresectable melanoma expressing BRAF V600E in Combination with trametinib for the treatment of unresectable or metastatic melanoma withBRAF V600EK in Combination with trametinib for the treatment of metastatic NSCLC with BRAF V600E in Combination with trametinib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with trametinib for the treatment of anaplastic thyroid cancer ATC that cannotbe removed by surgery or has spread to other parts of the body with BRAF V600E in Combination with binimetinib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Combination with cetuximab EGFR Ab for the treatment of metastatic colorectal cancerwith BRAF V600E in PLX8394BGB283TAK580Phase IIINCT02428712PLX8394 with cobicistat CYP3A inhibitor was well tolerated and showed promising activityin BRAFmutated refractory cancersPhase I NCT02610361Phase IIINCT03905148Evaluation of safety and PK of BGB283 alone and combination with mirdametinibPhase I NCT02327169Phase I NCT03429803TAK580 is the inhibitor of BRAF V600E and dimersTreatment in pediatric lowgrade gliomaCCT3833Phase I NCT02437227 CCT3833 is a panRAF inhibitor of mutant BRAF CRAF and SRC kinasesRAFMEKRO5126766Phase I NCT00773526Phase I NCT03681483Phase I NCT03875820Phase I NCT02407509RO5126766 is a dual inhibitor for both RAF and MEKTreatment of advanced KRasmutant lung adenocarcinomasEvaluation of safety and PK of RO5126766 with VS6063 FAK inhibitor or everolimusmTOR inhibitorRO5126766 showed activity across Ras and RAFmutated malignancies with significantresponse in lung and gynecological cancersMEK12TrametinibApprovedCobimetinib ApprovedPhase IIINCT03989115BinimetinibApprovedSelumetinibApprovedMirdametinib Phase IINCT03962543Phase IINCT02022982Phase IIINCT03905148A singleagent oral treatment for unresectable or metastatic melanoma with BRAFV600EK in Combination with dabrafenib for the treatment of unresectable or metastatic melanomawith BRAF V600EK in Combination with dabrafenib for the treatment of metastatic NSCLC with BRAF V600E in Combination with dabrafenib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with dabrafenib for the treatment of ATC that cannot be removed by surgeryor has spread to other parts of the body with BRAF V600E in In combination with vemurafenib to treat advanced melanoma with BRAF V600EK in Doseescalation of combination of RMC4630 SHP2 inhibitor and cobimetinibCombination with encorafenib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Selumetinib was approved for neurofibromatosis type with symptomatic inoperable plexiformneurofibromas according to NCT01362803Evaluation of mirdametinib in the treatment of symptomatic inoperableneurofibromatosis type1 NF1associated plexiform neurofibromas PNsCombination of mirdametinib with palbociclib in the treatment of KRasmutant nonsmall cell lung cancer NSCLCEvaluation of safety and PK of BGB283 alone and combination with mirdametinibSHR7390Phase I NCT02968485 Evaluation of safety and PK of SHR7390 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancers ContinuedTargetDescriptionEvaluation of safety and PK of CS3006CompoundCS3006Development stagesPhase I NCT03516123Phase I NCT03736850ERK12UlixertinibMK8353LY3214996ASTX029ATG017KO947Phase IIINCT01781429Phase I NCT04145297Phase IINCT03698994Phase I NCT03454035Phase I NCT01358331Phase I NCT03745989Phase I NCT02972034Phase I NCT04081259Phase I NCT04391595Phase I NCT02857270Phase IINCT04386057Phase IIINCT03520075Responses to ulixertinib in NRas BRAF V600 and nonV600 BRAF mutant cancersEvaluation of ulixertinib alone or combined with hydroxychloroquine palbociclibCDK46 inhibitor in MAPK mutated cancersMK8353 was optimized from SCH772984 for better pharmacokinetics and exhibitedinhibition of BRAF V600 mutant cancersEvaluation of combination of MK8353 with selumetinib or pembrolizumab PD1 Abin advanced malignanciesEvaluation of treatment of MK8353 alone or combined with abemaciclibCDK46 inhibitorHydroxychloroquine in advanced malignanciesEvaluation of safety and PK of ASTX029Phase I NCT04305249 Evaluation of safety and PK of ATG017Phase I NCT03051035 Evaluation of safety and PK of KO947dual inhibitor RO5126766 has been developed and exhibited a strong potential against both Ras and RAFmutated cancers in phase I clinical trials [] Thisallosteric inhibitor docks on MEK and prevents the release of MEK from RAF as well as the subsequent phosphorylation of MEK by RAF [] which gives it muchmore advantages than all other known RAF inhibitorsaccording to the regulatory mechanism of the RAFMEKERK kinase cascade [] As to small molecule inhibitors that target the terminal kinase ERK although anumber of them have been developed and are undergoing clinical trials [ ] their therapeutic values fortreating RasRAFmutated cancers remain unknownLike MEK inhibitorsthese ERK inhibitors may notachieve a good therapeutic index as single agents byvirtue of their inhibitory role in both malignant and normal tissues However they may contribute to antiRasRAF cancer therapy as synergetic agents combined withRasRAF inhibitorsOveralltargeting hyperactive MAPK signaling hasachieved exciting outcomes for treating RasRAFmutated cancers However although some effective smallmolecule inhibitors have been developed and applied toclinical treatment drug resistance and side effects remain remarkable challenges and there is still a long wayto develop a longeffective approach with manageableside effects for treating RasRAFmutated cancersAlthough hyperactive MAPK signaling has a dominantrole in cancer biology it is finetuned by other signalingssuch as PI3KAKTmTORC and AMPK during diseaseprogression [] These signaling interplays have important impacts on both cancer progression and clinicaltreatment based on MAPK inhibition In this review wewill focus on the crosstalk between MAPK and AMPKsignalingsAMPK signaling and its roles in cancer biologyAMPK signaling and cellular metabolismAMPK AMPactivated protein kinase is an energy sensorthat monitors the AMPADPATP ratio in eukaryotic cellsThis atypical protein kinase was firstly discovered as acontaminant during the purification of acetylCoA carboxylase ACC a wellstudied substrate of AMPK for fattyacid FA synthesis nowadays [] Fig Howeverthe phosphorylation of ACC by AMPK in response to thehigh AMPATP ratio had not been revealed until a decadelater [] and the enzyme was thus named as AMPKthereafter [] Fig Biochemical studies have shownthat AMPK consists of three subunits including the catalytic α subunit and the regulatory β and γ subunits [] Fig In mammals AMPK subunits are encoded asseveral isoforms α1 α2 β1 β2 γ1 γ2 γ3 which arepreferentially expressed in specific tissues or anisms[ ] For instance the α2 subunit associatesonly with β1 in type I muscle fibers while it binds to bothβ1 and β2 in type II muscle fibers [ ] Also theliver formulation of AMPK subunits differs among speciesas that α1β2γ1 is dominant in human whereas α1β1γ1and α2β1γ1 in dog and rat respectively [] Althoughan isoform replacement of AMPK subunits may not extensively affect the basal activity of AMPK as adaptive reit alters AMPKssponses such as exercise do []subcellular locations and sensitivity as well as interactionswith other signaling pathways [] The anismtissue 0cYuan Journal of Hematology Oncology Page of Fig Target hyperactive RasRAFMEKERK MAPK signaling for cancer therapy The RasRAFMEKERK MAPK signaling functions downstream ofreceptor tyrosine kinases RTKs Upon engagement by their ligands RTKs activates guanine exchange factors Sos proteins which load GTP to RasGTPases Then GTPbound Ras GTPases recruit RAFMEK heterodimers in cytosol to plasma membrane where they form transient tetramers throughthe sidetoside dimerization of RAFs The RAF dimerization not only turns on RAFs but also loosens RAFMEK heterodimerization and facilitates MEKhomodimerization on RAF dimer surface which leads to the activation of MEKs by RAFs Once MEKs are activated they phosphorylate ERKs and thenactive ERKs phosphorylate a number of downstream effectors In cancer cells hyperactive RasRAFMEKERK MAPK signaling arising from geneticmutations of Ras GTPases and BRAF can be targeted by small molecular inhibitors of Ras G12C BRAFV600E MEK and ERKstagespecific selectivity of subunit isoforms complicatesAMPKs regulationAs a key sensor of cellular energy stress the activity ofAMPK is predominantly regulated by cellular AMPADPATP that competitively binds to the γ subunit of AMPKand thus promotes or inhibits the phosphorylation ofThr172 on α subunit by the tumor suppressor liver kinaseB1 LKB1 or the dephosphorylation of this site by phosphatases [ ] Fig Besides adenine nucleotidesintracellular calcium ions activate AMPK through calciumcalmodulindependent protein kinase kinase CAMKK2 also called CAMKKβFig which acts downstream of the hormoneactivated receptors such as muscarinic receptors and ghrelin receptor onendothelial cells or neuron cells [] On the otherhand AMPK can be inhibited by a metabolite of glucosefructose 16bisphosphate FBP which binds to the aldolase and prevents the interaction of AMPK with LKB1 inglucoserich environments [] Fig Active AMPK[]has more than downstream substrates that regulatethe metabolism of lipids cholesterol carbohydrates andamino acidsActive AMPK promotes the oxidation of fatty acidsand inhibits the synthesis of fatty acids and cholesterolwhich involves largely in acetylCoA AMPK phosphorylates and inhibits HMGCoA reductase HMGR that requires acetylCoA in its reduction reaction [ ] Fig Also AMPK phosphorylates ACC that converts acetylCoA to malonylCoA and therefore slowsdown the de novo fatty acid FA synthesis and increasesthe FA oxidation [] Fig Alternatively AMPK regulates the lipid metabolism through altering the mitochondria structure and function In the mitochondriaAMPK phosphorylates Akinase anchoring protein AKAP1 a key scaffold protein for protein kinase APKA and facilitates the phosphorylation of a mitochondriafusion factor dynaminrelated protein DRP1 by PKA which promotes mitochondrial fusion 0cYuan Journal of Hematology Oncology Page of Fig AMPK signaling and its downstream effectors AMPK is activated by liver kinase B1 LKB1 or calciumcalmodulindependent protein kinasekinase CAMKK2β through phosphorylation on Thr172 of α subunit and is inactivated through dephosphorylation of this site by proteinphosphatases in response to changes of cellular AMPADPATP ratio Downstream effectors activated by AMPK are indicated as arrows and thoseinhibited by AMPK are shown as barheaded linesand oxidative phosphorylation [] Moreover AMPKaccelerates the mitochondria biogenesis likely throughphosphorylating and activating the transcriptional activator proliferatoractivated receptor gamma coactivator1alpha PGC1α [ ] Fig However upon energy stress AMPK plays an opposite role in mitochondria biology Under this condition AMPK is essential forthe fragmentation of mitochondria AMPK phosphorylates mitochondrial fission factor MFF on Ser129 andthereby facilitates the translocation of DRP1 from cytosol to mitochondria membrane in energy stressdrivenmitochondria fission [ ] Then AMPK promotesthe clearance of damaged mitochondria through autophagy In this process AMPK binds directly to and phosphorylates the unc51like autophagy activating kinase ULK1 Autophagyrelated gene ATG9 and Beclin which triggers the autophagosome formation [] Fig Active AMPK directly regulates the carbohydrate metabolism or indirectly through altering the fatty acid metabolism as described above Activation of AMPKstimulates the expression and plasma membrane translocation of solute carrier family member GLUT proteins and thereby facilitates glucose import [ ] Fig Intracellularly AMPK phosphorylates andactivatesis6phosphofructo2kinasePFK2thatfructose 26bisphoresponsible for the synthesis ofsphate a potent stimulator of glycolysis and thus accelerates glycolysis []Fig Furthermore AMPKappears to phosphorylate and inhibit glycogen synthasein the liver which dampens glycogen synthesis and thusindirectly enhances glycolysis []Active AMPK maintains cellular amino acid homeostasis mainly by controlling the activity of mammaliantarget ofrapamycin complex mTORC1 ThemTORC1 is a central sensor of cellular amino acids thatsamples amino acids in both cytosol and lysosome [] Upon activation by amino acids mTORC1 stimulates protein synthesis by phosphorylating ribosomalprotein S6 kinase B1 S6K and eukaryotic translationinitiation factor 4E binding protein 4EBP1 whichenhances the consumption of cellular amino acidsMoreover active mTORC1 blocks cellular autophagy byphosphorylating ULK1 and impairs the recycling ofamino acids [] Both effects of mTORC1 lead to a remarkable drop of cellular amino acid reservoir ActiveAMPK has been shown to inhibitthe activity ofmTORC1 direct and indirectly upon energy stresswhich limits the expenditure of amino acids Alternatively active AMPK can restrict protein synthesis byphosphorylating and thereby inhibiting eukaryotic translation elongation factor eEF2 kinase a key regulator 0cYuan Journal of Hematology Oncology Page of of protein synthesis [] To restore cellular amino acidreservoir active AMPK stimulates cellular autophagy asdiscussed above which degrades surplus or dysfunctional proteins into amino acids [] In addition it isworth noted that cellular amino acids can affect theactivity of AMPK reversely Dependent on conditionscontexts either amino acids may inhibit or stimulate theactivity of AMPK though underlying molecular mechanisms remain ambiguous []YAPactivity which impairsAMPK signaling in cancer biologyIt is well known that AMPK is a putative substrate oftumor suppressor LKB1 [ ] Fig Therefore AMPK has been generally considered as a key effector that mediates the tumorsuppressive function ofLKB1 Indeed a genetic ablation of the AMPK α subunitin mice accelerates Mycdriven lymphomagenesis throughfacilitating a metabolic shift to aerobic glycolysis []Simultaneously AMPK inhibitorsAMPKi promoteepithelialtomesenchymal transition EMT in breast andprostate cancers [] These studies validate AMPK as atumor suppressor under certain circumstances Furthermechanistic studies have demonstrated that AMPK prevents cancers through phosphorylating multiple targetsthat play indispensable roles on different layers of diseaseprogression AMPK phosphorylates angiomotin like AMOTL1 an adaptor protein in the HippoYap pathway and thus blocks Yes1 associated transcriptional regucellslatorproliferation and survival [] AMPK also phosphorylates TSC complex subunit TSC2 and regulatory associated protein of MTOR complex Raptor and therebyinactivates mTORC1 [ ] which in turn elevatescellular autophagy activity and inhibits cancer initiationTo bypass this inhibitory effect cancer cells can activatethe MAGE family member A MAGEA36tripartitemotif containing TRIM28 ubiquitin ligase complexthat targets the AMPK α subunit for degradation and thusreactivates mTORC1 to restrict cellular autophagy []Moreover AMPK is able to phosphorylate enhancer ofzeste polycomb repressive complex subunit EZH2and thereby disrupts the polycomb repressive complex PRC2 which relieves the epigenetic silence of tumorsuppressors in cancers [] Alternatively AMPK phosphorylates and stabilizes another epigenetic master regulator Tet methylcytosine dioxygenase TET2 whichfunctions as a putative tumor suppressor to preventtumorigenesis [] Altogether these findings indicatethat AMPK has a pronounced antitumor activity as itsupstream kinase LKB1 doescancerkillsandLICsstressleukemia TALL oncogenic Notch signaling induces ahigh level of aerobic glycolysis which needs to be restrained by AMPK and loss of AMPK results in energystressdriven apoptosis of leukemic cells and slows downdisease progression [] Similarlyin acute myeloidleukemia AML metabolic stress elevates the ROS leveland induces DNA damage in leukemiainitiating cellsLICs and AMPK confers metabolic stress resistance toLICs [] AMPK knockout or pharmaceutical inhibitionunder metabolicinhibitsleukemogenesis Moreover AMPK plays a determinantrole in maintaining the NADPH homeostasis in cancercells upon energy stress which is critic | 2 |
" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fishers exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolankis guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncolarjonasánchez a barrios p boldoroda e camps b carrascocampos jmartÃn vc garcÃafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629 xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial egypt journalof anaesth karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99 vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c" | 0 |
"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowneutrophils after capecitabine therapy had adverse clinical features Whats important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowneutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowpopulation might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [] Whats more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhats more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy []Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowmyeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15 neutrophils CD11bCD15CD14 or CD11bCD66bCD14and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhats important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16low group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was à Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by Ï2 test The data revealed that patients inCD16low group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16low group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16low group while only patients wereCD16 And patients with high CA199level were found in CD16low group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16low after therapy n nAll patients n nCD16 after therapy n nAge years¥GenderMaleFemaleTumor locationRectumColonTumor Size¥ cm cmCEA level after therapy¤ ngml ngmlCA199 level after therapy¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16low group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table Whatsimportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E Whats important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16low group p values were calculated by logrank test n in CD16 group and n in CD16low grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowmyeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowmyeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CIp valueMultivariate analysisHR95CIp value 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16myeloid cells we sorted peripheral blood CD11bCD16myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16 in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16N CRC R CD11bCD16myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearsons correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowneutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearsons correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowneutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients wont have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy Whats more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroups data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowmyeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e | 2 |
sarcomatoid carcinomas scs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology the features of this tumor were ï¬rst described in by snover scs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands however these tumors are rarein the digestive tract especially in the stomach as of april there are only six cases of gastric sarcomatoid carcinomagsc reported in the english medical literature these previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor due to the more invasive nature and poorerprognosis of gsc than pure gastric adenocarcinoma gacand gastric lymphoma gl it is clinically beneï¬cial to narrowdown the diï¬erential diagnoses by understanding the computedtomography ct characteristics of gsc the present studyanalyzed our experience in diagnosing ï¬ve patients with gscin terms of the imaging ï¬ndings and clinical features to thebest of our knowledge our study represents the largest seriesof gscs to datein addition due to the rarity of gsc the diï¬erential diagnosisbetween gsc and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of gsc from gac and glmaterials and methodsthe protocol was approved by the medical ethics committeeof zhengzhou universityinformed consent was obtainedfrom all patientspatient selectionfrom august to january we searched the pathologyrecords and the picture archiving and communication systemspacs of our hospital the search terms included stomachand sarcomatoid carcinomas a total of ï¬ve patients wereidentiï¬ed as having sc and were enrolled in the present study weretrospectively reviewed all clinical data demographic featureslaboratory ï¬ndings clinical interventions and the histologicï¬ndings of the ï¬ve biopsy or operation specimensct evaluationfive gsc patients underwent ct examinations the ctscans were acquired with a 64row multidetector devicediscoveryct750hd ge healthcare waukesha wiunited states conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgml ge medical systems mlkgand mls through a dualhead pump injector medradwarrendale pa united states the imaging parameters wereas follows tube voltage kv tube current ma ï¬eldof view fov mm matrix mm and sectionthickness mm finally a 20ml saline ï¬ush was performedat a rate of mlsgastric sarcomatoid carcinomacontrastenhanced ct scans were acquired with scanningdelays of s arterial phase ap and s portal venous phasepp after the iv injection of the contrast agent started the ctdose index volume for the three phases was msvimage analysistwo experienced radiologists and years of abdominal ctexperience performed a retrospective analysis of the ct imagesall analyses were performed with an aw47 workstation gehealthcare and the radiologists were blinded to the clinicalinformation of the patients the evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement the enhancement pattern of the tumor wasclassiï¬ed as homogeneous or heterogeneous based on the apimage the degree of enhancement of the tumor was based ondynamic ct imaging using hu attenuation where obviousenhancement was deï¬ned as hu moderate enhancementas hu and mildly enhancement as huthe gscs were staged with the union for internationalcancer control uicc tnm staging standard all imagingï¬ndings were compared with the postoperative pathologicalï¬ndings the accuracy rate the number of cts coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses pathological evaluationthree patients underwent gastrectomy and two underwentendoscopic biopsy the three gastrectomy specimens measured cm cm cm cm cm cmcm cm cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm cm cmand cm cm the remaining specimen was a soft massmeasuring cm cm cm for biopsy multiple sampleswere acquired and the diameter of each sample was cmaccording to the relevant literature the diagnostic criteria fsc were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue in addition if biopsywas performed the sarcomatoid component can be seen in everysample furthermore sarcomatoid regions express epithelialmarkers such as ck or emathe specimens were fully stretched ï¬xed and soaked in formaldehyde solution for h all biopsy specimenswere analyzed the specimens underwent routine dehydrationparaï¬n embedding sectioning into µm thick sectionsand hematoxylin eosin he staining immunohistochemicalstaining was performed using a roche benchmark xt automaticimmunohistochemical detector the antibodies used in thisstudy included ae1ae3 ckl ck818 epithelial membraneantigen ema vimentin p40 p63 and antigen ki67 ki67all antibodies listed above were purchased from dako dakoglostrup denmarkfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable comparison between gsc and gac glage median age rangemain symptomsepigastric discomfortpainintermittent vomitingacute hematemesisbloody stooldysphagialocationcardia and fundusbodyantrumthe longaxis diameter median size rangeshapefocal thickeningdiffuse thickeningmassserosal surfacebare areaclearunclearulcersyesnodensity characteristicsheterogeneoushomogeneousenhancement patterheterogeneoushomogeneouslymph node involvementyesnoliver involvementyesnotherapyresectionchemotherapyresection and chemotherapyneoadjuvant chemotherapyradiation therapygscgacgl years years years cm cm cm comprehensive comparative analysiseach patient with gsc was matched by age ± years year ofdiagnosis and sex to four patients with gac gl patients witheach disease were retrieved from pacs patients with gsc werecompared with those with gac gl in terms of demographicclinical and ct characteristics table resultspatient characteristicsthe patients included four men and one woman ranging inage from to years with a median age of years theclinical and ct features of these patients are summarized intables all patients had nonspeciï¬c symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingthe other presenting symptoms included hematemesis or weightloss three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery and two patients chose to deny treatment inaddition we also reviewed the upper gastrointestinal radiographyresults figure the laboratory ï¬ndings revealed that patient was positivefor tumor abnormal protein tap and patient was positivefor carbohydrate antigen ca125 before treatmenthemoglobin and erythrocyte count decreased in three patientsfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and pathological factors of the ï¬ve gsc patientscasesexage yearscomplaintlocationmaximumdiametertumor markercmanemiatherapymetastasismmfmmsuddenhematemesisepigastricdiscomfortintermittentvomitingepigastric painepigastric painepigastric painlesser curvatureremnant stomachcardia andfunduscardia and fundusfunduscardia and fundusnormaltap ca125 normalnarrnnonenonerc yespresentpositive noabsentnegative f female m male age in years r radical gastrectomy rn remnant gastrectomy c chemotherapyna not availabletable computed tomography features of the ï¬ve gsc patientscasegross features of the tumorulcersgrowth modedensity characteristicsenhancement patterfocal thickeningfocal thickeningmassfocal thickeningfocal thickeningintracavityintracavityintracavityintracavityintracavityheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousmarginunclearunclearunclearclearunclear yespresentpositive noabsentnegativefigure characteristics of xray examinations of a 65yearold male patient with gsc ab reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines the surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and pathological featuresmicropathologically the gastric tumor cells showed inï¬ltrativegrowth the cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged mitotic ï¬gures were visiblefigures 2ab on immunohistochemical examination thetumor cells showed positive staining for ae1ae3 cklck818 ema p40 vimentin the ki67 index was higher than figures 2ci all ï¬ve tumors were diagnosed as gscin addition the sarcomatoid component showed spindle cellsarcomatoid morphologyct findingsof the cases of gsc were in the gastric fundus and cardiafigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach the ct manifestations of this tumor included localthickening n mass formation n the longaxisfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure histological and immunohistochemical features of gsc ab hematoxylineosin he staining showing tumor cells demonstrated spindleshapedstructures signiï¬cant atypical nuclei pleomorphic nuclei and giant nuclei mitotic ï¬gures visible tumor cells showed inï¬ltrative growth cells were stained withhematoxylin and eosin stain magniï¬cation a b by immunohistochemistry the tumor cells were positive for ae1ae3 c ckl d ck818 e emaf p40 g and vimentin h moreover of them were positive for ki67 i the ï¬nal diagnosis was sc [magniï¬cation ci ]diameter of the lesions ranged from to cm mean size cm in addition ulcers with an irregular base and slightlyraised borders were observed in of cases among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaamong the two patients with biopsyproven gsc one patientexhibited tumor invasion of the gastric bare area the majorchanges in the ct imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat initially the ct ï¬ndings were interpreted as gac in fourcases and gl in the tumor showed predominantly inhomogeneous densityand the radiodensity values were hu in the noncontrastphase heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement the radiodensity values on the apimages ranged from to hu and to hu in thevenous phase after contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase one of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was conï¬rmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement the remaining patientshowed noevidence of metastasis among the two patients with biopsyspecimens one patient was identiï¬ed as having lymph nodemetastasis on ctlobe ofct staging versus pathological stagingof gscnone of the gscs were staged as t1t2 by ct or pathologythe accuracy of ct staging t3 and t4 gsc was and respectively the overall diagnostic accuracy of ctfor determining the t stage of gsc was none of the gscs were staged as n2n3 by ct or pathologythe accuracy of ct in staging n3 and n4 gsc was and respectively the overall diagnostic accuracy of ct fordetermining the n stage of gsc was the comparison of tn staging based on ct and pathology isshown in table frontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure sarcomatoid carcinoma of the stomach in 62yearold women a unenhanced ct image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region bd contrastenhanced ct image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase in perigastric lymph nodes an enlarged and signiï¬cantly enhancement lymph node can be seenb arterial phase of contrast enhancement image c portal phase of contrast enhancement image d portal phase of contrast enhancement coronal imagediscussiontable ct and pathological tn staging for comparisonsarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells however the spindle cells of scsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a speciï¬c line of mesenchymalcharacteristicsdiï¬erentiation moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved in the present study our patientstumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofsarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur in the digestive system the incidencesof scs in the esophagus and liver are relatively high but scsare exceedingly rare in the stomach we could ï¬nd only sixprevious reports in the english literature table between and patients with sc conï¬rmed by pathologywere retrospectively analyzed with only ï¬ve tumors occurringin the stomach the average age of the reported patientswas years range and that in our series was years range a previous study reported that the sexcaseno no no no no ctt4an0t3n0t3n1t3n0t4an1na not available t tumor n nodepathological staget4an1t3n0nanat4an0distribution of male to female gsc patients was and thecorresponding proportion in our patients was ithas been noticed that scs are more common in male patientsand sex is a probable risk factor gsc patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation due to thickening of the gastricwall pain or discomfort in the upper abdomen is common thesymptoms can last from a few days to several years withoutobvious speciï¬cityin the present study of the cases of gsc were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach four cases of gsc in the present study hadfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and imaging features of six previously reported cases of gcscasegenderageyearslocationsize cmshapeulcersenhanceappearancerecurrencemetastasistherapyprognosis mmffmmremnant stomachgreater curvaturelesser curvaturegastroesophageal junctionremnant stomachdistal stomachpolypoidpolypoidpolypoidulceratedpolypoidmassnenenenenehypernenonesurgerysurgerysurgeryendoscopysurgeryna mo d mo d mo d mo d mo dthe patient succumbed to heart failure before the surgical treatment an autopsy was performed yespresentpositive noabsentnegative hyper hyperdensene no evaluate mo month d dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmthe location distribution and longaxis diameters of the gscs inour patients were similar to those in previous reports in the actual diagnosis processthe diagnosis of sc has always been diï¬cult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsthe sarcoma components show typical specialized diï¬erentiation howeverthe termssarcomatoid carcinoma and carcinosarcoma have been usedinterchangeably in some cases therefore the understanding ofthese tumors has been hampered nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective the ct ï¬nding sc in the stomach have notbeen previously scientiï¬c reported or even detailed descriptionthere are only four simple descriptions chunchao reported that a patient with a giant sc presented a mass witha cm diameter in the antrum and body of stomach whichinï¬ltrated the gastric serosa the hepatic ï¬exure of the colon andgallbladder were also involved on ct contrastenhanced ctimages showed obvious enhancement of the two lesions sato reported a patient with sc of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter the other two reports only describeda soft tissue mass or a large tumor in the dilated stomach on the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than sc gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in of all reviewed cases tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion enhanced abdominalct showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes yoshiyuki reported a 70yearoldjapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andct revealed an ulcer on the lesion the contrastenhanced ctimages showed heterogeneous enhancement of the mass theï¬nal pathological diagnosis was gastric carcinosarcoma inthe present study we found that gsc showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers the site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach the signal of the tumor was homogeneousor heterogeneous on plain ct scans after contrast mediuminjection of tumors demonstrated heterogeneousenhancement on ap images due to cystic areas or necrosis inthe lesions in this study the enhancement degree of all tumorsreached a peak in the pp after contrast enhancement for thesetumors the enhancement degree in the delayed phase wasnot signiï¬cantly reduced the overall enhancement mode wasdelayed enhancement in addition ct showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue these ï¬ndingsreï¬ect the metastatic and highly invasive characteristics of gscoverall ct and contrastenhanced ct can clearly show theprimary lesion inï¬ltration range lymph node metastasis anddistant metastasis of gsctomographic diagnosis of gsc has not been attemptedbecause of the rarity of this entity according to the ï¬ndingsof our study gsc needs to be diï¬erentiated from gac andgl on ct adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach the incidenceof gac is high in men and the median patient age is years the most common ct signs of gac are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer tumors involving the mucosal surface can appearenhanced s after injecting a contrast agent the peakvalue for tumors invading the muscular layer usually appearsafter s and after the mucosal surface is strengthenedthe duration is longer primary gl accounts for ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus the incidenceof gl is high among males with a median patient age of yearsthe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue the most common ct manifestations ofgl are diï¬use thickening of the gastric wall or a homogeneousfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall for gl becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on ct gl originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage after contrast medium injection most gl showedhomogeneous and slight enhancement in the delayed phase lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis the ct ï¬ndings may only reï¬ect features of gsc but cannotaccurately diagnose gsc l one explore the origin of thesarcomatous portion immunohistochemistry ihc also failedto conclusively establish the origin of gsc rodrigues usedï¬uorescence in situ hybridization fish to conï¬rm that sc andadenocarcinoma have a common origin that is the epithelium while primary gl originated from gastric submucosallymphoid tissuethe main treatment for localized lymphomas is eradicationof helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone surgery is the only treatment option for patients with gacadjuvant chemotherapy and chemoradiotherapy are also oftenused targeted therapy isin the exploration stage however there are currently no speciï¬c national comprehensivecancer network guidelines for the treatment of only gscbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method for examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for gsc and the eï¬cacy ofchemotherapy remain controversial domblides ï¬rstevaluated the eï¬cacy of immune checkpoint inhibitors icis forsc and found that lung sc patients exhibited high response ratesand prolonged overall survival os with icis this studyprovides a new idea for the treatment of gscbecause gl tends to be conï¬ned to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of gac previous literature has found that scin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months similarly gscpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at theï¬rst diagnosis all these clinical manifestations suggest that gschas a poorer prognosis than gac and gl in additiongsc can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver this conclusion is consistent with ourresearch resultsconclusionthe incidence rate of gsc is extremely low so clinicians andradiologists are not familiar with the features of this tumorbased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that gsc is morecommon in men who are approximately years old and isoften accompanied by ulcers the disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced ctimages these characteristics can provide a reference for furtherresearch on gscs in the future however an accurate diagnosisof gsc depends on the combination of clinical imaging andhistopathological features due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with ct are essentialdata availability statementthe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorethics statementthe studies involving human participants were reviewed andapproved by the medical ethical committee of the zhengzhouuniversity the patientsparticipants provided their writteninformed consent to participate in this study written informedconsent was obtained from the individuals for the publication ofany potentially identiï¬able images or data included in this author contributionsyl manuscript preparationliterature research and dataanalysis pl literature research and data analysis kf manuscriptreview and data collection kc guidance of pathologicalknowledge sy guidance of imaging knowledge jj imaging datacollection and analysis wl and xz manuscript editing jgstudy conception and design manuscript review and guarantor ofintegrity of the entire study all authors have read and approvedthe ï¬nal manuscriptfundingthis work was supported by the national natural and sciencefund of china no references zhu cc li mr lin tl zhao g sarcomatoid carcinoma of the stomach acase report and literature review oncol lett doi ol20153460 snover dc levine gd rosaij thymic carcinoma five distinctivehistological variants am j surg pathol zhou dk gao bq zhang w qian xh ying lx wang wl sarcomatoidcarcinoma of the pancreas a case report world j clin cases doi 1012998wjccv7i2236 xie y xiang y zhang d yao x sheng j yang y sarcomatoidthe doi 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature mol med repafrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinoma sato a oki e kohso h endo y uchida h hiroshige s sarcomatoidcarcinoma of the remnant stomach report of a case surg today doi 101007s0059501204027 nakayama y murayama h iwasaki h iwanaga s kikuchi m ikeda s gastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation pathol int doi 101111j144018271997tb04540x robeycaï¬erty ss grignon dj ro jy cleary kr ayala ag ordonezng sarcomatoid carcinoma of the stomach a report of three caseswith immunohistochemical and ultrastructural observations cancer doi 101002109701421990040165730co2n ruess da kayser c neubauer j fichtnerfeigl s hopt ut wittel uacarcinosarcoma of the pancreas case report with comprehensive literaturereview pancreas doi 101097mpa0000000000000904 fujiie m yamamoto m taguchi k iwanaga a ohgaki k egashira a gastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review surg case rep doi 101186s407920160176z tanimura h furuta m carcinosarcoma of the stomach am j surg doi 101016000296106790325x kitamura s study on carcinosarcoma of stomach gan kumagai k kawai k kusano h matsuo k irie j tsuchiyama h a caseof socalled carcinosarcoma of the stomach gan no rinsho bekki t fujikuni n tanabe k yonehara s amano h noriyuki t thegastric carcinosarcoma with severe venous invasion a case report surg caserep doi 101186s4079201804218 ikeda y kosugi s nishikura k ohashi m kanda t kobayashi t gastriccarcinosarcoma presenting as a huge epigastric mass gastric cancer doi 101007s1012000604054 cid³n eu cuenca ij gastric adenocarcinoma is computed tomography ctuseful in preoperative staging clin med oncol doi cmos2641 hallinan jt venkatesh sk gastric carcinoma imaging diagnosis stagingand assessment of treatment response cancer imaging doi gossios k katsimbri p tsianos e ct features of gastric lymphoma eurradiol doi 101007s003300050069 rodrigues dn hazell s miranda s crespo m fisher c de bono js sarcomatoid carcinoma of the prostate erg ï¬uorescence insituhybridization conï¬rms epithelial origin histopathology doi 101111his12493 levine ms rubesin se pantongragbrown l buck jl herlinger h nonhodgkins lymphoma of the gastrointestinal tract radiographic ï¬ndings ajram j roentgenol doi 102214ajr16818976941 russo ae strong ve gastric cancer etiology and management in asia and thewest annu rev med doi 101146annurevmed081117 domblides c leroy k monnet i mazi¨res j barlesi f gounant v eï¬cacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma jthor oncol doi 101016jjtho202001014 niu x sarcomatoid carcinoma in the parotid gland a review of years ofexperience laryngoscope doi 101002lary27474 li s jiang l he q wei w wang y zhang x the prognostic signiï¬canceof jmjd3 in primary sarcomatoid carcinoma of the lung a rare subtypeof lung cancer onco targets ther doi 102147otts22 dai l fang q li p liu f zhang x oncologic outcomes of patients withsarcomatoid carcinoma of the hypopharynx front oncol doi103389fonc201900950 seo n kim mj rhee h hepatic sarcomatoid carcinoma magnetic resonanceimaging evaluation by using the liver imaging reporting and data system eurradiol doi 101007s00330019060528 shi y chen j chen h hong x sarcomatoid carcinoma of the gallbladdera case report j int med res doi conï¬ict of interest the authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestcopyright liu liang feng chen yue ji li zhao and gao this is anopenaccess distributed under the terms of the creative commons attributionlicense cc by the use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice no use distribution or reproduction is permitted which does not complywith these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | 0 |
"Supplementary Material Supplementary Data Click here for additional data file. Determination of SETDB1 gene amplification and its association with RNA and protein overexpression in lung cancer cell lines. (a) Assessment of SETDB1 copy-number by quantitative genomic PCR. Amplification frequency of SETDB1 (evaluated with SYBR Green Bio-Rad Hercules CA USA) was calculated by the standard curve method using the 7900HT SDS program. To define an internal control gene we chose chromosome 1p36.23 because it is the least aneuploid region among our cell lines (PEX19 gene). Primers are available upon request. DNA from normal lung was used as the reference standard. Results are reported as n-fold copy-number increase relative to the PEX19 gene. (b) Fluorescence in situ hybridization for the SETDB1 gene. The UCSC genome browser (http://www.genome.ucsc.edu) was used to select the bacterial artificial chromosome (BAC) clone spanning the 1q21 region for the SETDB1 gene: RP11-42A12. A telomeric BAC clone located in the telomeric 1p36.23 region was used as a control. The BACs were obtained from the BACPAC Resource Center at the Children's Hospital Oakland Research Institute (Oakland CA USA). SETDB1 and telomeric probes were labeled with Spectrum Green and Red dUTP (Abbott Wiesbaden Germany) respectively using a CGH Nick Translation Reagent Kit (Abbott Molecular Inc. Des Plaines IL USA). The samples were counterstained with 4'6-diamidino-2-phenylindole in Vectashield antifade solution (Burlingame CA USA). Gene amplification was observed in the interphases of NCI-H1437 NCI-H1395 and DMS-273. Probes were verified to give a single signal on normal commercial lymphocyte metaphase slides (CGH Reagents Abbott). Quantitative reverse transcriptionPCR (c) and western blot (d) demonstrate higher levels of SETDB1 mRNA and protein (ab12317 Abcam Cambridge UK) respectively in amplified cancer cell lines (H1437 NCI-H1395 and DMS-273) than that in unamplified cells. PCR primers are available upon request. Growth-promoting effects of SETDB1 in lung cancer. (a) Stable downregulation of the SETDB1 gene by short hairpins using two different target sequences for DMS-273 (clones A30/A31 and clone B32-63) and NCI-H1437 (clones A56-B and B46-9). SETDB1 shRNA sequences are available upon request. (b) The short hairpin SETDB1-depleted cells were less viable in the 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide (MTT) assay than in the untransfected or scrambled shRNA-transfected cells (P-values obtained by the analysis of variance (ANOVA) test). (c) The colony formation assay showed that DMS-273 and NCI-H1437 cells stably transfected with the shRNA against SETDB1 formed significantly fewer colonies than scrambled shRNA-transfected cells (P-values obtained by the ANOVA test). Data shown are means±s.d. n=3. (d) Effect of SETDB1 shRNA-mediated depletion on the growth of DMS-273 and NCI-H1437 xenografts in nude mice. Tumor volume was monitored over time and the tumor was excised and weighed at 30 days. There was a significant decrease in tumor weight in the SETDB1 shRNA-stably transfected cells (P-values obtained by the ANOVA test). Data shown are means±s.d. n=10. Impact of SETDB1 on invasiveness and chemosensitivity. (a) Effect of SETDB1 on the invasion potential of A549 cells determined by the matrigel invasion assay. Cells were transfected with 3??g of Flag-SETDB1 or empty vector in 60?mm dishes. After 24?h cells were stimulated or not with phorbol myristate acetate (PMA) plus ionomycin (Io) for 30?min. Then cells were trypsinized and 5 104 cells were resuspended in serum-free media and added to the upper compartment of a transwell coated with 1?mg/ml Matrigel (BD Biosciences Lexington KY USA). Media with 10% fetal bovine serum was added in the lower compartment and cells were incubated at 37?°C for 42?h. Invasive cells were fixed with phosphate-buffered saline 4% paraformaldehide stained with 0.5% violet crystal and visualized and photographed under a 10 magnification objective with a microscope. Invasive cells were counted using ImageJ 1.45s (Wayne Rasband National Institutes of Health Bethesda MD USA) and percentage of invasive cells were represented. Results are the mean of at least three experiments by duplicate and the significance was determined using analysis of variance test. *<P=0.05. (b) Cancer cells harboring the SETDB1 gene amplification are sensitive to the decrease in cell viability caused by mithramycin a SETDB1-interfering drug. 3-(45-dimethyl-2-thiazolyl)-25-diphenyl-2H-tetrazolium bromide (MTT) assays in control-scrambled shRNA DMS-273-transfected cells in comparison with three shRNA-stable downregulated SETDB1 clones (A21 A30 and A31) show enhanced inhibition of viability in cells with SETDB1 gene amplification-mediated overexpression. Detection of SETDB1 gene amplification and its associated overexpression in primary tumors from lung cancer patients. (a) Fluorescence in situ hybridization for the SETDB1 gene shows gene amplification in the primary lung tumors 1 2 and 3. SETDB1 unamplified tumors are shown in the cases 4 and 5. The UCSC genome browser (http://www.genome.ucsc.edu) was used to select the bacterial artificial chromosome (BAC) clone RP11-42A12 spanning the 1q21 region of SETDB1 gene. A telomeric BAC clone located in the telomeric 1p36.23 region was used as a control. (b) Immunohistochemistry for SETDB1 (HPA018142 Sigma-Aldrich St Louis MO USA) shows overexpression of the protein in the above shown three primary lung tumors harboring SETDB1 gene amplification. Minimal expression is detected in the unamplified cases (4 and 5). Magnification 100. (c) Association between SETDB1 gene amplification and overexpression in the studied fifty-nine cases is shown. Fisher's test two-tailed P-value<0.0001. 0135054 6265 Oncology Oncology Oncology 0030-2414 1423-0232 24925190 4105702 10.1159/000360703 NIHMS595992 Distribution and timing of distant metastasis after local therapy in large cohort of patients with esophageal and esophagogastric junction cancer Shiozaki Hironori Sudo Kazuki Xiao Lianchun Wadhwa Roopma Elimova Elena Hofstetter Wayne L. Skinner Heath D. Lee Jeffrey H. Weston Brian Bhutani Manoop S. Blum Mariela A. Maru Dipen M. Ajani Jaffer A. U. T. M. D. Anderson Cancer Center (UTMDACC) Houston Texas USA All correspondence to: Jaffer A. Ajani Department of Gastrointestinal Medical Oncology University of Texas M. D. Anderson Cancer Center 1515 Holcombe Blvd (FC10.3022) Houston TX 77030 jajanimdanderson.; Phone: 713-792-2828; Fax: 793-745-1163 13 6 2014 07 6 2014 2014 07 6 2015 86 0 336 339 Background Patients with localized esophageal and esophagogastric junction cancer (EAC) receive chemoradiation then surgery (trimodality; TMT) or definitive chemoradiation (bimodality; BMT). Since distant metastases (DMs) are common but the details of the DM distribution and timing in a large cohort have not been described. Methods 629 patients with localized EAC who had TMT or BMT were analyzed. Standard statistical methods were used to define the endpoints. Results The median follow-up time was 37.2 months (interquartile range: 17.865.0). Among 356 TMT patients 33% (119) developed DM as their first relapse and among 273 patients with BMT 40% (109) developed DM. 91% (TMT) and 96% (BMT) of DMs were diagnosed within 2 years of local therapy. The most common sites of DMs were: lung distant nodes liver peritoneal cavity bone brain and pleura in the order of frequency. The median overall survival of TMT patients with DM was 10.2 months (95% CI: 7.812.7) and that for BMT patients with DM was 7.8 months (95% CI: 5.79.9). Conclusions Following TMT or BMT ?33% of patients developed DMs and most DM occurred within 2 years (>90%) of local therapy. A clinical model that highly associates with high-risk for DM in TMT-eligible patients prior to surgery is desirable. Risk of metastases Adenocarcinoma Chemoradiation Chemotherapy Esophageal cancer gastroesophageal cancer Metastasis Cell Death Dis Cell Death Dis Cell Death & Disease 2041-4889 Nature Publishing Group 24625970 3973193 cddis201456 10.1038/cddis.2014.56 Original Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer Antitumor activity of phenothiazines in SCLC Zong D 1 2 * Zielinska-Chomej K 1 Juntti T 1 M¶rk B 1 Lewensohn R 1 H¥¥g P 1 Viktorsson K 1 * 1Department of Oncology-Pathology Karolinska Biomics Center Karolinska Institutet Stockholm Sweden *Department of Oncology-Pathology Karolinska Biomics Center Karolinska Institutet Z5:01 S-171 76 Stockholm Sweden. Tel: +1 301 435 5764; Fax: +1 301 402 0711; E-mail: dali.zongnih.gov (DZ) or Tel: +46 8 517 701 77; Fax: +46 8 517 710 00; E-mail: kristina.viktorssonki.se (KV) 2 Current address: Laboratory of Genome Integrity National Cancer Institute National Institute of Health Bethesda MD USA 03 2014 13 03 2014 1 3 2014 5 3 e1111 24 09 2013 23 12 2013 14 01 2014 Copyright © 2014 Macmillan Publishers Limited 2014 Macmillan Publishers Limited This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-nd/3.0/ Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies especially new targeted therapies. In this work we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease however extended cell line analyses as well as in vivo studies are needed to make such conclusion. small cell lung cancer phenothiazines lysosomal dysfunctions Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders for example schizophrenia.1 In this setting the therapeutically relevant targets of phenothiazines are believed to be neurotransmitter receptors in particular the dopamine D2 receptor (D2R).2 However an array of putative cellular targets have additionally been described for phenothiazines ranging from calmodulin (CaM) to lipid membranes suggesting that the effect of these compounds likely extend beyond their impact on neuroendocrine signaling.3 Notably various studies have demonstrated that phenothiazines possess cytotoxic activities especially in established tumor cell lines.4 However there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death and numerous hypotheses including CaM antagonism5 membrane fluidization6 and disruption of mitosis7 have been proposed. Lung cancer (LC) remains one of the most deadly malignancies worldwide8 despite considerable effort in the development of treatment strategies especially new targeted therapies. In parallel there has been a recent revival of interest in the use of old' FDA-approved drugs for new indications. In this work we evaluated the potential utility of phenothiazines in LC. We show that phenothiazines induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. In this context cytotoxicity was due to phenothiazine-induced lysosomal dysfunction and the higher susceptibility of SCLC cells correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward the conventional chemotherapeutic agents for example cisplatin etoposide or gemcitabine. Our data thus uncovered a novel context-dependent antitumor action of phenothiazines in SCLC. Results Single-drug treatment with phenothiazines induced cytotoxicity preferentially in human SCLCs Phenothiazines have been reported to induce apoptosis in tumor cells of different origin.34 9 Here we evaluated the potential therapeutic utility of phenothiazines in a panel of human SCLC and NSCLC cell lines. At a concentration of 10??M which is in the range of concentration achievable in the plasma of patients without eliciting significant adverse side effects10 phenothiazines were overtly cytotoxic analyzed as reduction in cell viability in most of the tested SCLC cell lines while NSCLC cell lines were generally less responsive (Figures 1ac Table 1). The hyper-responsiveness of SCLC to phenothiazines was not recapitulated when the chemotherapeutic drugs cisplatin etoposide and gemcitabine were used11 as comparable decrease in cell viability was observed in SCLC and NSCLC cells (Supplementary Figure S1). Moreover our results clearly show that the sensitivity to phenothiazine-induced decrease in cell viability in SCLC is not substantially influenced by growth pattern site of isolation prior exposure to conventional chemotherapies and occurs despite mutation in the tumor suppressor p53 (Supplementary Table S1). To further verify the preferential activity of phenothiazines in SCLC we compared the sensitivity of four phenothiazine compounds (trifluoperazine dihydrochloride (TFP) fluphenazine dihydrochloride (FPZ) triflupromazine hydrochloride (TFPZ) and promazine hydrochloride (PZ)) over a range of concentrations in the SCLC cell line H82 and in the NCSLC cell line U-1810 (b upper panel). We also assessed the dose-dependent cytotoxicity of TFP analyzed as decrease in cell viability in several additional SCLC (H69 U-1285 U-1906 and U-2020) and NSCLC (A549 H125 and H1299) cell lines (b lower panel). These analyses confirmed that SCLC cells were more sensitive to phenothiazines than NSCLC cells (b). Importantly the cell viability of primary fetal lung WI-38 fibroblast was less affected by phenothiazines at concentrations that caused significant cytotoxicity in SCLC cells illustrating a potential therapeutic window for phenothiazines in SCLC (Figures 1a and c). The increased sensitivity of SCLC cells to phenothiazines was also evidenced by increased cell death and growth arrest after treatment with these agents (). Thus 10??M TFP induced a prominent impairment in cell division capacity in SCLC cells while in NSCLC cells 20??M TFP was needed to significantly affect proliferation (Figures 2a and b). Analysis of cell cycle kinetics of SCLC cells (H69 and H82) revealed that 10??M TFP caused a time-dependent cell cycle arrest in late S and G2/M phases whereas such changes in cell cycle were not observed for NSCLC (U-1810; c). Taken together our data demonstrate that phenothiazines preferentially decrease the viability and growth of human SCLC." | 1 |
" ovarian cancer is the leading cause of cancerrelated death among women complete cytoreductivesurgery followed by platinumtaxene chemotherapy has been the gold standard for a long time variouscompounds have been assessed in an attempt to combine them with conventional chemotherapy to improvesurvival rates or even overcome chemoresistance many studies have shown that an antidiabetic drug metforminhas cytotoxic activity in different cancer models however the synergism of metformin as a neoadjuvant formulaplus chemotherapy in clinical trials and basic studies remains unclear for ovarian cancermethods we applied two clinical databases to survey metformin use and ovarian cancer survival rate the cancergenome atlas dataset an l1000 microarray with gene set enrichment analysis gsea analysis western blotanalysis and an animal model were used to study the activity of the aktmtor pathway in response to thesynergistic effects of neoadjuvant metformin combined with chemotherapyresults we found that ovarian cancer patients treated with metformin had significantly longer overall survival thanpatients treated without metformin the protein profile induced by low concentration metformin in ovarian cancerpredominantly involved the aktmtor pathway in combination with chemotherapy the neoadjuvant metforminprotocol showed beneficial synergistic effects in vitro and in vivos this study shows that neoadjuvant metformin at clinically relevant dosages is efficacious in treatingovarian cancer and the results can be used to guide clinical trialskeywords neoadjuvant metformin ovarian cancer clinically relevant dosage aktmtor pathway synergisticeffects correspondence syeungmdanderson limh33mailsysueducn kuochang wen and pilin sung share first authorship9department of emergency medicine division of internal medicine theuniversity of texas md anderson cancer center houston tx usa10guangdong research institute of gastroenterology the sixth affiliatedhospital of sun yatsen university yuancun erheng rd guangzhou pr chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwen of ovarian research page of ovarian cancer is the fifth leading cause of mortality in developed countries in the united states an estimated women were diagnosed with ovarian cancer in and deaths due to ovarian cancer occurred complete cytoreductive surgery followed by standard firstline platinumtaxene chemotherapy has been shown to improve the survival rate however the majority of patientsexperience relapse and the 5year survival rate is approximately chemoresistance to platinumbased treatment remains a major challenge in the successful treatmentof ovarian cancer and the mechanisms underlying platinum resistance are multifactorial various cellular processes are observed in resistant cells and activation of thepi3kakt pathway is believed to be a determinant of resistance in ovarian cancer [ ] thus the development ofan improved treatment to overcome acquired resistance incancer cells or decrease the side effects of platinumbasedtreatment is needed to treat ovarian cancermetformin n0n0dimethylbiguanide a biguanide is anoral hypoglycemia agent that is widely used as an antidiabeticdrug to treat type diabetes mellitus dm it is also widelyused to treat polycystic ovarian syndrome metformin hasbeen shown to reduce cancer development in type dm patients and inhibit growth in several cancer models [ ] either alone or in combination with cytotoxic agents [ ]the major target of metformin in cancer cells is the tumorsuppressor lkb1ampactivated protein kinase ampkpathway which serves as a metabolic checkpoint to arrestcell growth when intracellular atp levels are low such as innutrientpoor conditions after activating ampk metformin phosphorylates tuberous sclerosis complex tsc2and then binds with its obligate partner tsc1 tsc2 leadsto the accumulation of rhebgdp and the inhibition ofmtorc1 which influence eukaryotic translation initiationfactor 4ebinding protein 4ebp1 and ribosomal s6 kinases6k1 respectively shank showed that metformincan restrict the growth and proliferation of ovarian cancerstem cells yasmeen revealed that metformininduces apoptosis in ovarian cancer cell lines in an ampkindependent manner by activating caspases downregulating bcl2 and bclxl expression and upregulating baxand bad expression which results in cell cycle arrest in the sand g2m phases rattan identified metformin asan antiproliferative therapeutic that can act through bothampkdependent and independent pathways via thesepathways metformin inhibited cell proliferation in bothwildtype and ampk null mouse embryo fibroblasts as wellas in ampksilenced ovarian cancer cells in addition metformin has been shown to inhibit pi3kaktmtor signaling in lung cancer [ ] breast cancer pancreaticcancer and hepatic cancer however most studies showing that metformin alleviates cancer have used higher doses in vitro than thoseused in diabetic patients these high concentrationsmay directly cause the death of tumor cells in thepresent study we tested a low concentration as the effective dose which was a clinically relevant dose the effects of lowconcentration metformin on aktmtorsignaling in ovarian cancer remain unclearthe aim of the present study was to examine the effectsof a combination of metformin at clinically relevant dosages and chemotherapy on ovarian cancer via the aktmtor pathway we found that metformin reduced ovarian cancer death in two clinical datasets and predicted thatthe effect of metformin in ovarian cancer was mediated bythe aktmtor pathway using a bioinformatics modelthen we demonstrated that the low concentration ofmetformin inhibited the growth of a mouse ovarian surface epithelial cell line mosec and that it had a synergistic effect in combination with chemotherapy via theaktmtor pathway neoadjuvant application of metformin plus chemotherapy yielded beneficial synergistic effects both in vitro and in vivo the results provide insightinto the potential of neoadjuvant metformin to augmentthe efficacy of existing cancer therapeuticsresultsthe effect of metformin on survival in ovarian cancerpatientswe investigated the impact of metformin on humanovarian cancer by analyzing a clinical dataset in total patients were diagnosed with primary ovarian cancerin the department of gynecology and obstetrics taipeiveteran general hospital from to after theirclinical and drug histories were reviewed patientswho underwent complete surgery and were treated withcarboplatin were included in the analysis thirtytwo ofthese patients took metformin either during admissionor in the outpatient clinic os was measured from thedate of diagnosis to death or was censored at the date ofthe last followup the os of patients with metformintreatment n was significantly higher than that ofpatients without metformin n p fig 1atable figure 1b shows the ovarian cancerfree incidence of female dm patients n fromthe national health insurance taiwanese dataset ovarian cancer was less frequent among metformininsuthan among metforminlin users n insulin users n p the use of metformin or insulin may help prevent ovarian cancer in female dm patients we next investigated the cellulareffects of metformin on ovarian cancer the expressionprofiles of differentially expressed genes in response totreatment with metformin chemotherapy or both wereretrieved from the l1000 study gsea was performedusing the up and down gene expression datasets froml1000 gsea revealed that the gene expression induced 0cwen of ovarian research page of fig the effect of metformin on survival in ovarian cancer patients a kaplanmeier os of ovarian cancer patients with n or without n metformin use b the ovarian cancerfree incidence in female dm patients metformin ever usedinsulin ever used users n and metformin never usedinsulin never used users n from the national health insurance taiwanese dataset c tumorproliferation cell number 2dcolony formation and western blot analyses of the indicated proteins in the aktmtor pathway of mouse ovariancancer cells treated with different concentrations of metformin d comparison of the mortality rates between groups with low and high proteinexpression by the halfdivision approach kaplanmeier analysis assessed the correlations of the indicated proteins akt [total and pser473] mtor[total and pser2448] with the overall survival of patients data from the cbioportal tcga database tcga provisional ovarian cancer genomicsn a logrank pvalue less than indicated a significant difference in overall prognosis p p p by control and metformin treatment was similar to thatof the kegg pancreatic cancer pathway httpwwwgenomejpdbgetbinwww_bgetpathwayhsa05212fig s1 which predominantly involved the aktmtorpathway based on the proteomic and viability investigation metformin may involve the alternation of phosphorylation in the aktmtor pathway accompanyingcell retardation in ovarian cancer without affecting thetotal amount of protein fig 1c metformin inhibitedthe aktmtor pathway in a dosedependent manneras shown by western blot analysis the phenomenonwas found at both high mm and low mmdoses of metforminlow doses were closer to theclinically relevant concentration fig s2 at low dosesof metformin the alternation of phosphoampk wasnot as obvious as that in the aktmtor pathway wefurther investigated the clinical role of the aktmtorpathway using the tcga ovarian cancer dataset thepatients with upregulated phosphorprotein akt_pser473 or mtor_pser2448 had significantly poor osthe expression of total protein was not associated withclinicalimportance in ovarian cancer fig 1d thesefindings highlight the value of metformin in inhibitingovarian tumor cells via phosphorylation of the aktmtor pathway which indeed plays an essential role inthe prognosis of ovarian cancer 0cwen of ovarian research page of table the clinical characteristics of the study populationcharacteristicp valueno metformin usen metformin usen diabetes ratestageaearly i ii late iii iv histologybepithelialother typesc ± ± ± ± ca125ddeath rateoverall survivale§afigo stage international federation of gynecology and obstetrics surgicalstaging of ovarian cancer missing value not included in statistical testbmissing value not included in statistical testcincluding primary peritoneal serous carcinoma ppscdbefore surgery mean ± sd umleoverall survival months mean ± sdcalculated by chisquare testcalculated by students ttest§calculated by kaplanmeier logrank testmetformin at a clinically relevant dosage inhibits ovariancancer growth through the aktmtor pathwayto mimic the clinically relevant dosages in the humanbody we used lowdose concentrations of metformin inthe study we assessed the cell growth of metformin in mmtreated ovarian cancer cell lines to evaluate thegrowthinhibitory effect of metformin as shown infig 2ab mm metformin can reduce colony formation and cell growth both mouse and human ovariancancer cell lines manifested significantly reduced proliferation after treatment with clinically relevant doses ofmetformin fig s3a furthermore mm metformintreatment beginning from day to day reduced cellviability and cell viability recovered from day to day after discontinuing treatment fig 2c the inhibitory effect of lowdose metformin on the aktmtor pathwaywas shown during metformin treatment day to day the phosphoprotein expression was inhibited duringmetformin treatment but these expression levels wererestored when suspending metformin fig 2d theaforementioned data suggested that metformin at lowdose concentrations could inhibit cell growth in ovariancancer through inhibition of the aktmtor pathwayas supported by the gseathe synergistic effects of metformin and chemotherapyon ovarian canceralthough standard firstline platinumbased protocolsimprove survival in ovarian cancer strengthening thesechemotherapy regimens is warranted we evaluated theantiproliferative effects of different protocols beforeduring or after and doses of metformin in combinationwith chemotherapy fig 3aleft panel as shown infig 3a concurrent combination of metformin andchemotherapy yielded the strongest inhibition fortyeighthour exposure to concurrent metformin andchemotherapy resulted in a clear synergistic effect withnegative log ci values in ovarian cancer cells fig 3aright panel regarding human ovarian cancer cellslower concentrations of carboplatin μm but nothigher concentrations μm show synergy with metformin with negative log ci values fig s3b the activated forms of akt are key intracellular mediators ofgrowth cell survival and platinum response determining the activation state of the aktmtor pathway isimportant for understanding the synergistic mechanismof action of lowdose metformin combined with chemotherapy in ovarian cancer western blot analysis demonstrated that both chemotherapy alone and metforminalone reduced the levels of phosphorylated aktmtorwithout affecting the total amount of aktmtor protein fig 3b the combination of metformin and chemotherapy produced a stronger inhibition of pakt and theakt downstream effectors pmtor ser2448 ps6 kinaseser235236 and p4ebp1 thr3746 compared withmetformin or chemotherapy alone in contrast the totalamounts of mtor s6 kinase and 4ebp1 were unaffectedby treatment and ampk phosphorylation was not reduced by treatment with lowdose metformin or chemotherapy either alone or in combinationsome dm patients treated with metformin cannotachieve good blood sugar control and require other medications we investigated the cellular effects of metformintreatment on tumors in patients with poor blood sugarcontrol fig 3c left panel as shown in fig 3c middlepanel mtt assays indicated that cells cultured in highglucose medium mgl showed accelerated cell proliferation relative to that of cells in control medium mgl however metformin treatment diminished this effect induced by high glucose the combined effect of metformin and chemotherapy in a highglucose medium wasantagonistic with a positive log ci fig 3c right panelto determine whether high glucose induces the aktmtor pathway in mosecs we treated the cells with metformin alone chemotherapy alone or a combination inhighglucose medium or control medium for h fig s4highglucose medium resulted in marked increases inpakt pmtor ps6 kinase and p4ebp1 levels comparedwith the control medium treatment with metformin andcarboplatin in the highglucose medium reduced the protein level of pakt but not the protein levels of pmtorps6 kinase and p4ebp1 relative to the levels in the controlmedium these results revealed that high glucose or poorblood sugar control may diminish the antitumor effects ofmetformin and chemotherapy although they still have effects on the paktmtor pathway 0cwen of ovarian research page of fig see legend on next page 0cwen of ovarian research page of see figure on previous pagefig metformin at a clinically relevant dosage inhibits ovarian cancer growth through the aktmtor pathway ab growth of mouse ovariancancer cell line mosec in cells incubated for serial days with lowconcentration metformin mm met metformin p by twowayanova c and d cell viability and protein analyses of the aktmtor pathway under treatment with metformin mm from day to day andwhen treatment was suspended from day to day the beneficial synergistic effects of neoadjuvantmetformin under combination treatmentwe further investigated the synergistic effects of lowconcentrations of metformin and chemotherapy underdifferent treatment regimens commonly used in clinicalsettings as shown in fig 4a synergistic effects were obviously observed in both protocol neoadjuvant metformin and protocol concurrent metformin but notin protocol adjuvant metformin western blot analysis indicated that protocols reduced the activitiesof pakt pmtor ps6 kinase and p4ebp1 with protocols and having superior effects compared withprotocol fig 4b these results indicate that metformin should be used before or at least concurrent withchemotherapy to enhance the antitumor effect neoadjuvant metformin combined with chemotherapy was betterthan the combination protocolwe further investigated the in vivo antitumor activityof neoadjuvant metformin in b6 mice bearing mosecsthat were grown subcutaneously as tumor xenograftstreatment with metformin or chemotherapy as singleagents caused a decrease in tumor size relative to that ofcontrol untreated mice treatment with the neoadjuvantmetformin combined with chemotherapy significantlyreduced tumor growth fig 4c finally we selected thecases with chemotherapy from fig 1a to determine theclinical impact of metformin in chemotherapy metformin before or during chemotherapy indeed showed atrend toward better overall survival compared with single chemotherapy although this trend did not reach statistical significance fig 4dschematics of the intracellular effects of treatmentwith metformin chemotherapy or their combination onovarian cancer are shown in fig 4e neoadjuvant metformin combined with chemotherapy produced bettersynergistic effects inhibiting akt and its downstreampathway a highglucose environment such as that inpoorly controlled type dm patients may increase activated aktmtor signaling thusfor patients withpoor glucose control the combination of metformin andchemotherapy may be slightly superior to chemotherapyalone and not as efficacious as that in patients with goodglucose controldiscussiondiabetes is strongly associated with an increased incidence of cancer many studies have shown thatmetformin can reduce the risk of cancerincludingbreast colon liver and pancreatic cancers and improveoutcomes over those obtained with other antidiabetictreatments sulfonylurea insulin in diabetic patients whether metformin can reduce the risk of ovarian cancer has been investigated [] but few studies havefocused on the effects of metformin combined withcommonly used firstline chemotherapeutic drugs suchas carboplatin and the underlying mechanisms neoadjuvant metformin combined with other therapieshave been administered to treat erpositive breast cancer in a phase ii clinical trial clinicaltrialsgov identifier nct01589367 in this study we evaluated thesynergistic effects of neoadjuvant metformin combinedwith chemotherapy in ovarian cancer via the aktmtor pathway both in vitro and in vivo and found thatpoor glucose control diminished the synergistic antitumor effects of combination treatmenta previous casecontrol study that used the ukbasedgeneral practice research database revealed thatthe adjusted odds ratio of metformin use vs nonuse forovarian cancer incidence was not significant in nondiabetic patients but was significant in diabetic patientsin another recent metaanalysis which included one observational study and two clinical trials the pooled oddsratio ci of metformin use for ovarian cancer incidence was recently a study wasperformed using reimbursement databases of the national health insurance nhi to evaluate metforminuse in taiwanese women with type dm metformindecreased the incidence of ovarian cancer and the overall fully adjusted hazard ratio ci for everusersversus neverusers was in thepresent study the results were similar to those of a previous report in which ovarian cancer was less likelyto occur in metformininsulin users n than in metformininsulin users n thisprevious study similarly used reimbursement databasesof the nhi although the data were from a different timeperiod the hospital cohort data showed that ovariancancer patients treated with metformin had a significantly longer os compared with patients not treatedwith metformin this finding is similar to that of another casecontrol studyin which an association between metformin use and improved survival of ovariancancer patients was identified these results indicated that metformin may reduce ovarian cancer incidence in type dm patients and improve survival aftera diagnosis of ovarian cancer 0cwen of ovarian research page of fig the synergistic effects of metformin and chemotherapy on ovarian cancer a synergistic effects of metformin combined withchemotherapy carboplatin at different concentrations and different combination protocols concentration of metformin and mmconcentration of carboplatin and μm met metformin chemo chemotherapy b the effects of metformin alone mmchemotherapy carboplatin μm alone or combined treatment assessed by the indicated antibodies in western blot analysis c mtt assaysshowed that cells cultured in a highglucose medium mgl exhibited greater growth than those in control medium mgl greensquares represent cells cultured in highglucose medium and red solid circles represent those cultured in control medium concentration ofmetformin and mm concentration of carboplatin and μmmost studies examining the effects of metformin oncancer have used doses mm higher than those usedclinically for diabetic patients [ ] yielding metforminplasma concentrations between and μmoll fewstudies have focused on the impact of metformin at clinically relevant dosages although dr hu and colleagues 0cwen of ovarian research page of fig see legend on next page 0cwen of ovarian research page of see figure on previous pagefig the beneficial synergistic effects of neoadjuvant metformin under combination treatment a and b protocol a neoadjuvant protocolmosecs treated with metformin alone for day and then with a combination of metformin and carboplatin for days protocol a concurrentprotocol mosecs treated with both metformin and carboplatin from day for days protocol an adjuvant protocol mosecs treated withcarboplatin alone from day for day and then with a combination of metformin and carboplatin for day pink circles represent log ci valuesunder protocol green squares represent those under protocol and yellow triangles represent those under protocol concentration ofmetformin and mm concentration of carboplatin and μm met metformin chemo chemotherapy imagej analysis for relativeintensity of protein bands c mosecs were injected into b6 mice n which were divided into groups and subcutaneous tumor size wasmeasured after different treatments control metformin or carboplatin alone neoadjuvant metformin from monday combined with carboplatinfrom wednesday subcutaneous tumors were assessed at the end of the experiment control vs met p control vs chemo andmet chemo p d kaplanmeier os of ovarian cancer patients with n or without n metformin in before or duringchemotherapy e the model of synergistic inhibitory effects by neoadjuvant metformin combined with chemotherapy in the aktmtor pathwayadministered lowdose metformin mm or mmto suppress ovarian and breast cancer cell growth and survival in vitro the aim of their study was to investigatewhether lowdose metformin reprograms these cancercells into noncancerous cells in a foxo3dependentmanner which may allow patients to successfully overcome these cancers with minimal side effects howeverthey did not compare the inhibitory effects or therapyprotocol of metformin combined with chemotherapy inaddition they administered metformin via intravenous injection of metformin mgkg bw in mice which contrasts the more common oral route used in clinicalapplications for example diabetes mellitus in thepresent study we tested a low concentration of mm μmoll as the effective dose the cellular events observed in vitro suggest that this dose is safe and can betranslated to in vivo conditions the dosage of metformingiven to mice was mgkgday which is equivalent to mgday for a 60kg person according to a formulasuggested by the national institute of health usa this equivalent dosage is times lower than the maximum safe dosage of mgday recommended in thephysicians desk referencemetformin activates ampk via lkb1 which leads tothe inhibition of mtor signaling and its major downstream effectors the 4ebps and p70s6ks and the inhibition of global protein synthesis and proliferation invarious cancer cell lines [ ] in previous studies metformin tested on ovarian cancer was found to induce cellcycle arrest apoptosis angiogenesis and decreasedpmtor expression p38 mapk pathway activity and cancer stem cell activity in this study weshowed for the first time the effects of metformin on geneexpression patterns using gesa and the l1000 systemand found that the effects of metformin on gene expression in ovarian cancer are similar to those following activation of the kegg pancreatic cancer pathway fig s1in previousfollowing metformin treatmentphosphoakt levels decreased in two pancreatic cancercell lines a549 and panc1 we analyzed tcga dataand found that the phosphoaktmtor pathway is a determinant of clinical survival in ovarian cancer [ ] westudiesalso demonstrated for the first time the effects of metformin in combination with carboplatin a firstline chemotherapy drug and showed that the synergism of thesedrugs is due to the inhibition of the aktmtor pathwaywhich is independent of ampk at micromolar concentrations of metformin mm these results are consistentwith those of rattan who showed that metformin as an antiproliferative therapeutic can act throughboth ampkdependent and independent pathways metformin can be a veryveryvery inexpensive drug compared with aktmtor inhibitors and it may also haveantitumor effects knowledge of this mechanism may beuseful in clinical trials to adjust the dosage of chemotherapy or further overcome the chemoresistance to platinumin the futuremetformin can be added at different times beforeduring and after adjuvant chemotherapy and the effectsof these addition time points need to be investigated inthe present study a synergistic effect ci was observed in the neoadjuvant protocol and concurrentprotocol protocols but not in protocol adjuvantneoadjuvant metformin protocol was better than theconcurrent regimen these results are similar to those oferices although they did not assess the adjuvant use of metformin the optimal time frame of neoadjuvant metformin before chemotherapy should bedetermined in future studies or clinical trials althoughwe observed an inhibitory effect of neoadjuvant metformin day prior to chemotherapy in vitro and daysprior to chemotherapy in vivo furthermore the ovariancancer patients have a better prognosis with metformincombined with before or during chemotherapy compared with chemotherapy alone due to the small casenumber and retrospective analysis our results are notsignificant the optimal time frame for neoadjuvant metformin administration before chemotherapy will allow forthe acquisition of the most ideal results in basic experiments and clinical analyses these findings provide beneficial evidence that can guide the design of clinical trialsthe differences among protocols observed in this studymay be related to the cytotoxic effects of metformin oncancer stem cells which can enhance the efficacy of 0cwen of ovarian research page of neoadjuvant and concurrent chemotherapy by preventingthe establishment of chemoresistant clonesreducesome patients treated with metformin continue to showpoor blood sugar control and high blood glucose may diminish the antitumor effect of metformin karnevi reported that metformin can significantlytheproliferation of several pancreatic cancer cell lines under normal glucose conditions however they found that hyperglycemia reduced metformininduced growth inhibition byenhancing the igfi response and activating akt whichstimulated ampkser485 phosphorylation and impairedampkthr172 zhuang obtained similar results inbreast cancer and ovarian cancer cell lines reporting that cancer cells became less responsive to metformin when glucosewas increased to mm in a breast cancer cell line underlowglucose conditions metformin significantly decreased thephosphorylation of akt and various targets of mtorwhereas phosphoampk was not significantly altered in thepresent study we used an ovarian cancer cell line and demonstrated that a highglucose medium decreased the response tometformin the synergistic effects of carboplatin and metformin were abolished the phosphorylation of akt in lowglucose conditions mgl was substantially reduced bymetformin and phosphorylation levels of targets of mtors6k and 4ebp1 were decreased relative to those in highglucose conditions mm in ovarian cancer phosphoampk was not significantly altered the response to metformin was substantially altered in lowglucose conditions basedon previous studies and our observations we hypothesize thathigh glucose fuels glycolytic metabolism which maintains cellular atp levels when metformin blocks mitochondrial function when glucose is limiting cancer cells lack sufficient fuelto maintain glycolytic metabolism additionally mtor signaling is blocked in an ampkindependent manner enhancingmetabolic deficiency cellular atp is depleted leading to energy collapse and cell death sin lowconcentration metformin treatment ofpatients with ovarian cancer may have antitumor effectsand synergistic effects when used in combination withchemotherapy through the aktmtor pathway neoadjuvant metformin is a more preferable protocol than theconcurrent regimen future prospective clinical trials inpatients with ovarian cancer are required to investigatethe beneficial effects of neoadjuvant metformin in augmenting the efficacy of existing cancer therapeuticsmethodspatient samples from taipei veteran general hospitalmedical centera total of patients were diagnosed with primaryovarian cancer in the department of gynecology andobstetrics taipei veteran general hospital from to after a review of the patients clinical and drughistories patients who underwent complete surgeryand were treated with platinumbased therapy plus paclitaxel were included in the analysis of these patients were identified as having taken metformin eitherduring admission or in the outpatient clinic the overallsurvival os was measured from the date of diagnosisto death or was censored at the date of the last followup all documents were collected under protocols approved by the institutional review board of the hospitalpatient samples from the national health insurancetaiwanese datasetthe reimbursement data of taiwanese female patientswith a new diagnosis of type dm between and n were retrieved from the nationalhealth insurance database among these patients noneused only insulin or only metformin therefore we compared two groups those who received metforminand insulin n and those who received neiinsulin n then wether metformin norfollowed the two groups for newly diagnosed ovariancancer from to thirtyseven patients acrossthe two groups were diagnosed with ovarian cancermicroarray analysisthe microarray experiments were conducted following thel1000 operating procedure l1000 sop briefly thehuman ovarian cancer cell line es2 was left untreated control or treated with micromolar concentrations of metformin mm μm carboplatin or a combination in amicroplate after h of drug treatment the medium was removed and lysis buffer was added included in the l1000kit to the wells for min after cell lysis the lysate wasstored at °c for at least one night before being transferred to a 384well plate which was performed using theprotocol avai | 0 |
Circulating tumor cells CTCs derived from primary tumors andor metastatic tumors aremarkers for tumor prognosis and can also be used to monitor therapeutic efï¬cacy andtumor recurrence Circulating tumor cells enrichment and screening can be automatedbut the ï¬nal counting of CTCs currently requires manualintervention This not onlyrequires the participation of experienced pathologists but also easily causes artiï¬cialmisjudgment Medicalimage recognition based on machine learning can effectivelyreduce the workload and improve the level of automation So we use machinelearning to identify CTCs First we collected the CTC test results of patientsAfter immunoï¬uorescence staining each picture presented a positive CTC cell nucleusand several negative controls The images of CTCs were then segmented by imagedenoising image ï¬ltering edge detection image expansion and contraction techniquesusing pythons CV scheme Subsequently traditional image recognition methodsand machine learning were used to identify CTCs Machine learning algorithms areimplemented using convolutional neural network deep learning networks for trainingWe took cells from patients for training and testing About cells wereused for training and the others were used for testing The sensitivity and speciï¬city ofrecognition reached and respectively We will further revise our modelshoping to achieve a higher sensitivity and speciï¬cityKeywords circulating tumor cells CTCs imFISH machine learning image segmentation CNN networkINTRODUCTIONThe metastasis of cancers is a complex and multistage process The circulating tumor cells CTCsare the seeds shed from the primary tumor andor metastatic lesions and rooted in a new soiltransferred by the circulatory system Paget Circulating tumor cell is an intermediate stageof cancer metastasis correlated with cancer aggressiveness and the likelihood of metastasis andtherefore can be used to predict disease progression and survival on a realtime basis by liquidbiopsy Lindsay Praharaj Anand and Roszik Baek Maly Marcuello Pan Riebensahm The molecular subtypesof CTCs not only the CTCs count are interrelated with the prognosis BanysPaluchowski Cristofanilli Dong Stefanovic Whats more the PDL1Edited byCheng GuoColumbia University United StatesReviewed byJuanying XieShaanxi Normal University ChinaKhanh N Q LeTaipei Medical University TaiwanCorrespondenceBinsheng Hehbscsmu163comQiliang Zhou13974942986163comYuebin LiangliangybgeneiscnGeng Tiantianggeneiscn These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in Bioengineering andBiotechnologyReceived March Accepted July Published August CitationHe B Lu Q Lang J Yu HPeng C Bing P Li S Zhou Q Liang Yand Tian G A New Methodfor CTC Images Recognition Basedon Machine LearningFront Bioeng Biotechnol 103389fbioe202000897Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine Learningexpression in CTCsis correlated with the response toimmunity inhibitors Kloten PDL1EMTCTCs were associated with significantly poorer survival aftercurative surgery showing that PDL1 expression and EpithelialMesenchymal Transition EMT of CTCs are negative survivalpredictors for Nonsmall celllung cancer NSCLC patientsJanning Manjunath Pretreatment PDL1 CTCs are usually associated with a bad prognosis in patientstreated with PD1 inhibitors in NSCLC such as nivolumabGuibert The liquid biopsies worked as an ongoing monitoring systemto assess tumor heterogeneity and make it possible to detect asingle CTC or clusters of cells Wan Merker Praharaj Asante The breakthroughfor CTCdetection is the application of immunomagnetic CTCenrichment combined with ï¬ow cytometry which is still thegold standard of CTCdetection Racila Howeverthis method that lack of the cancer speciï¬c markers still remainslots of limitation Grover Ferreira Gabriel Keller Thusthe multimarker immunoï¬uorescence staining is required for recognizeCTCs Antibodies against chromosome centromere duplicationCEP8chromosome centromere duplication CEP17 areused to mark the rapidly dividing tumor cells antibodies againstCD45 as typical leukocytes ï¬laments as well as 4cid486diamidino2phenylindole DAPI for labeling nuclears Koudelakova Lu Liu Lee Although there are great advantages in enrichment technologythe automatic recognition of CTCs still remains problemsManual identiï¬cation is very timeconsuming and unreliableWith the continuous deepening of the application of CTCsrecognition in various cancer diseases the demand for rapidand automatic identiï¬cation and counting methods of CTCs isincreasing Several studies have reported the automated screeningprocess Nagrath Yang Kraeft performed a ï¬uorescencebased automated microscope systemREIS for cell detection This scanning can quantify the numberof cells reliably and reproducibly and categorize positive cellsbased on the marker expression proï¬le Ligthart redeï¬ned the CTCs by computer algorithms after the manualcounting The stricter deï¬nition with the standard deviationof the signal in the CKPE channel the peak signal value inboth the DNADAPI and CD45APC channels and the size ofthe objects used as classiï¬er was well validated CTC by clinicaloutcome using a perfectly reproducing automated algorithmMingxing reported an automated CTC enumerationZhou Allimages with diï¬erent colors weretransferred to a grayscale image and the grayscale images wereused to identify the position and outline of cells Howeverdespite the widely accepted these classiï¬cation methods stillremain subjective as the rules are set artiï¬cially The ï¬xedconditions may not identify the morphologically heterogeneousCTCs integrally Whats more diï¬erent technologies usually usediï¬erent antibodies making comparison and standardizationacross diï¬erent platforms challenging Marcuello With the maturity of artiï¬cial intelligence AI recent yearsmachine learning become an exciting ï¬eld for research TheUS Food and Drug Administration FDA has approved severalcommercial products using machinelearning algorithms in themedical diagnosis and research The cardiovascular MRI analysissoftware of Arterys was the worlds ï¬rst internet platform formedical imaging AI powered and FDA cleared This software isable to analyze multiple multiperiod MR images to determineblood ï¬ow in heart and main vessels The cloud platformwill enable software to collect and analyze the vast amount ofcardiovascular data from MR scanners in real time which willspeed up doctors diagnosis This artiï¬cial machine is consistentand tireless and is able to identify characters beyond humanperception which provided a substantial interest in the ï¬eldof medical research speciï¬cally medical images Dominguez Erickson Lundervold and Lundervold Maier Many algorithms are developed forselecting the best weights for features involving neural networksHornik decision trees Quinlan supportvector machines Cristianini and ShaweTaylor the na¯veBayes Lowd and Domingos knearest neighbors Zhouand Chen and deep learning McBee Wainberg Zou Deep learning as wellas deep neural network learning refers to the use of neuralnetworks with more than layers able to integrate vast datasetslearn arbitrarily complex relationships and incorporate existingknowledge Convolutional neural networks CNNs is a powerfulalgorithm for advancing biomedical image analysis as it assumesthat the input layer has a geometric relationship such as the rowsand columns of images Anthimopoulos Poplin It has been successfully applied in the cancer diagnosisand nuclei or tissue identiï¬cation Le Le Xing present a novel method for automatednucleus segmentation powered by CNNs The features involvedin the images are considered as a part of the search processand there is no need to limit the features compared to thetraditional machine learning methods which will eliminate thebias created subjective Here we apply deep learning to therecognition of CTCs in order to reduce the artiï¬cial errors andimprove accuracyMATERIALS AND METHODSPatients and Samples PreparationA cohort of patients with cancers were enrolled inthis study during which was approved by theethics committee of Chifeng Municipal Hospital The clinicalpathological characteristics of patients including age genderCTC number and cancer type are summarized in Table Fourmilliliter of peripheral venous blood was routinely collectedfor every patient The ï¬rst ml blood samples obtainedafter puncture was discarded in order to avoid the skinepithelial cells contamination Then the blood was placed inanticoagulation tubes and store at room temperature The testwas completed within hAll the patients were divided into two parts according tothe collecting date The earlier patients we collected wereused as the training data the others were used as the independentFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Clinical pathological characteristicsClinicopathologic variableAgeGenderSamples typeCTC numberCancer typeCategoryMeanMaleFemaleUnknownPeripheral bloodMeanLung cancerLiver cancerGastrointestinal cancerBreast cancerCarcinoma of thyroidNPCOtherClinical leveltesting data Thousand three hundred cells images in the earlierreceived patients were selected to build the CTC recognitionmodel which will be further tested by the cells images of thetest dataset There was no cross part between the two datasets inorder to avoiding the overï¬ttingEnrichment and imFISH Identiï¬cation ofCTCsThe Cyttel method was used to isolate and enumerate CTCsThe peripheral blood was ï¬rst centrifuged at g for minto get the precipitation and then washed by CS1 buï¬er CyttelBiosciences Co Ltd Beijing China Then the red blood cellswere lysed by CS2 buï¬er Cyttel After centrifuged at gfor min the precipitate was washed by CS1 buï¬er Thenthe cells were incubated completely with antiCD45 monoclonalantibodyconjugated beads Cyttel for min Three milliliterseparation medium was used to separate the beads and the CTCsby gradient centrifugation at g for min Then the upper rarecell layer was centrifuged at g for min and resuspendedby CS1 The tube was put on a magnetic stand for min Aftersmeared ï¬xed and dried cells were used to perform the imFISHThe slides were ï¬xed dehydrated and then dried at roomtemperature µl CEP8CEP17 antibody was added to the cellsand the slides were placed in a hybridization and denatured for h at ¦C The probe was eluted and the slides were washedtwice in SSC Then the CD45 ï¬uorescent antibody was addedto the sample area and the slides were put in a wet box andincubate for h at ¦C After incubation CD45 ï¬uorescentantibody was aspirated and µl mounting media containingDAPI was added to the sample area After mounted the cells canbe observed and counted under a ï¬uorescence microscopeThe Manual Interpretation Standard ofCTCs CountingAfter imFISHlots ofimages were acquired with diï¬erentï¬uorescent colors Usually manual counting is the goldstandard but its a time consuming and exhausted processionThe Manual interpretation standard of CTCs counting is Eliminates the aggregation superposition and interference ofnucleus or impurity DAPI positive CD45 negative and Three or more than three CEP8CEP17 signal points Itwill be regarded as one signal point if the distance between twosignal points is smaller than the diameter of one pointThe Image Segmentation Method WasUsed to Segment Single Nucleus andGive Labels of Cells Instead of ManualSince the obtained microscopic image is very huge the algorithmwill be limited by the memory and cannot be executed normallyon a conventional computer We ï¬rst selected part of the imagecontaining one CTC cell and several nonCTC cells around toperform the following test The chosen resolution is The CV package of python was used to process theCTCs images including conversion of color and morphologicaltransformations The RGB image was converted to the gray image The derivatives were calculated using the CVfunction Sobel from an image Morphological transformations operations based on theimage shapeThe Morphological package of python was used to segmentthe images of CTCs by image denoising image ï¬ltering edgedetection image expansion and contractionNuclei were segmented in the blue channel DAPI and theproportion of red in the red channel was detected based onthe position of the nucleus The nucleus with proportion of redhigher than was deï¬ned as having a common leukocyteantigen The orange channel was used to detect the number ofCEP8 chromosomes and the green channel was used to detectthe number of centromere probes extracted by CEP17 Diï¬erentcell types were distinguished by diï¬erent colors Figure The CNN Deep Learning Method WasUsed for CTCs Identiï¬cationWith the development of AI machine learning has been wildlyused in the procession of medical images Deep learning is a bigimprovement on artiï¬cial neural networks allowing higherlevelfeature extraction and better data prediction with more layersAfter segmentation CNN network were used to identify CTCcells in single nucleus Finally it enters the output layer andoutput the result ie CTCs or nonCTCsOur CNN model was built based on AlexNet which wasï¬rst introduced in Krizhevsky The networkconsists of eight weighted layers Figure the ï¬rst ï¬ve layersare convolution layers and the remaining three layers are fullconnection layers The output of the last full connection layeris the input of the dimensional softmax values which willgenerate the distribution network of two types of labelsThe ï¬vefold cross validation was used to prevent overï¬ttingand select hyperparameters of the model The best crossvalidation score was obtained by searching the hyperparameterspace round and round The ï¬nal hyperparameters involved inFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The imFISH result and the segmentation of chromosome and nuclear AC The imFISH result of CEP8 CD45 and DAPI D The merge of panelsAC E The CTCs were identiï¬ed by CV segmentation method and marked in red box ac The CEP8 signal points were identiï¬ed by CVsegmentation method and marked in red boxour model are activation function kernel regularizer type andregularization factor The workï¬ow is shown belowSp The grid was deï¬ned on 3dimensions with eachofthese maps for hyperparameter sets eg hyperparameters activation function kernel regularizer typeregularization factor activation function softmaxReLU tanh kernel regularizer type l1 l2regularization factor The range of possible values were deï¬ned of eachdimension Allestablishing the best onethe possibleconï¬gurations weresearched forEvaluation Criteria for Classiï¬cationModelsAfter segmentation some performance evaluation criteria Xie were involved in to evaluate the performance of theclassiï¬cation model such as sensitivity Se or recall speciï¬citySp precision F1 score and area under the receiver operatingcharacteristic curve AUCSerecall TPTP FNTNTN FPTPprecision TP FPF1 precision recallprecision recallIn the equations TP stands for the number of positive CTCcells which are correctly recognized as positive CTC cells FPstands for the number of negative CTC cells that are incorrectlyrecognized as positive CTC cells FN stands for the numberof positive CTC cells incorrectly recognized as negative CTCcells TN stands for the number of negative CTC cells correctlyrecognized as negative CTC cells Table RESULTSPatient CharacteristicsA total of patients were enrolled in this study from January to June The average age is years old Patients withlung cancer count of all patients and the next is breastcancer and gastrointestinal cancer Table Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The layers of the CNN model The ï¬rst ï¬ve layers are convolution layers and the remaining layers are full connection layersThree SubImages Were Required forManual CountingWe performed imFISH for all the patients and required images of CTCs cells Every image was divided into or channels with diï¬erent color The orange channel representedthe chromosome with CEP8 Figure 1A the green channelthecentromereofthechromosomerepresentedCEP17 Supplementary Figure S1represented the whitethe blueFigure 1C The mergence wasWe then manually labeled all withred channelcell with CD45 Figure 1Brepresented the nuclei with DAPIshown in Figure 1Dthese subimages accordingchannelFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Confusion matrix deï¬nitionsTABLE The confusion matrix of the models for test datasetConfusion MatrixPredictionMethodConfusion MatrixPredictionPositiveNegativePositiveNegativeTruePositiveNegativeTrue positive TPFalse positive FPFalse Negative FNTrue Negative TN CVALexNetTrueTruePositiveNegativePositiveNegativethetoare CTCs positivestandard AmongourresultspatientsTABLE Tuning of the hyperparameters of AlexNetActivation functionKernel regularizer typeRegularization factorThe Segmentation of Nuclear andIdentifying CTCs by CVSegmentation MethodIn order to avoid the artiï¬cial error and save costs we performedthe traditional image identiï¬cation method for CTCs countingFigure The nucleus was separated in the blue channelDAPI Figure 1E and the red proportion of the red channelwas detected according to the location of the cell nucleus Theproportion higher than was deï¬ned as the number of theCEP8 chromosome detected by the common antigen orangechannel of white blood cells Figures 1AC the number ofcentromeric probes detected by the green channel such as CEP17Supplementary Figure S1After segmentation of nuclear we used CV segmentationmethod to identify CTC cells from single nucleus regions in testing dataset by the manual interpretation standard ofCTCs counting After identiï¬cation and judgment cells of negative nuclei were recognized as CTC negative About cells of positive nuclei were recognized as CTCnegative The sensitivity and speciï¬city were and while the precision and F1 score reached and respectively Table We also applied the regionbased image segmentationalgorithm such as watershed algorithm in the segmentationprocess The watershed algorithm was implemented the bywatershed function in CV python and CV In this method optimal threshold value was used respectivelyin binaryzation process by setting THRESH_OTSU mode Thetraditional watershed algorithm was sensitive to noise and theaccuracy was lower than our segmentation method on CTCnegative data set in size of Supplementary Table S3The HyperParameters Selected forEvaluating the CNN MethodWe used GridSearchCV class in scikitlearn by providinga dictionary of hyperparameters to determine the hyperparameters of the model After the crossvalidation processactivation function was set to ReLU kernel regularizer type wasset to l2 and regularization factor was set to as shown inTable with the best performance Further the hyperparameterswe selected were used to construct the model on the wholetraining datasetsoftmaxReLUtanhl1l2l1l2l1l2The underline value shows the best result of AUC value in the tuning process of thehyperparameters of AlexNetThe Identiï¬cation of CTCs by CNNMethodWe got nuclei of patients by segmentation processFigure showed the whole ï¬owchart of the experiment About nuclei were used for training the left were usedfor testing We use the same images for testing cells of negative nuclei were recognized as CTC negative and cells of were recognized as CTC positive The sensitivityand speciï¬city were and while the precisionand F1 score reached and respectively Table and Figure Before that we also compared the performance of AlexNetmodel with others such as ResNet and Xception All ofthem have close AUC values Figure butthe AlexNetwas less timeconsuming in the training and test processSupplementary Table S1DISCUSSIONThis study showed a method for CTC counting powered bymachine learning The use of machine learning for imageinterpretation can capture important image features reduceerrors caused by manually setting interpretation standardsand save time and labor costs Although this method showsa higher sensitivity and speciï¬city in CTC countingisslightly worse than the ï¬rst method for the data used inthis study Actually we have analyzed that the main reasonis that there are fewer positive samples for training and thealgorithm cannot extract features of more positive samplesin the group were excludedIn additiondue to quality problems Unfortunatelythe CTC imagesincluded in the group doesnt cover the whole ï¬lm but asome picturesitFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The ï¬owchart of the whole experimentFIGURE The ROC curve of AlexNet ResNet and Xception modelpicture just focused on a certain CTCpositive cell under themicroscope which results in that the machine learning methodhas no advantage in recognition speed compared with thetraditional image recognition method Enlarging the scope ofimages and collected more samples is also that need to beimproved in the futureDeep learning has already been shown to be suitablefor detection of CTCs because of the high sensitivity andspeciï¬city in CTC counting We had changed the ï¬lter sizeand number in all convolution layers in order to ï¬nd thebest CNN parameters We found diï¬erent ï¬lter size andnumber will inï¬uence the results largely We changed ï¬lternumber from range to in our training process Wefound that the training result was not convergence when thenumber was less than It showed that the range of thefeature number of the image is about We tried toincrease the ï¬lter size from to but the result was notchanged a lot and the convergence speed even became slowerwhen the ï¬lter size higher than From this process wesummarized that the feature size in CTCs could not be greaterthan pixels Furthermore there are many appropriately AImodels such as VGG InceptionV14 We will apply themon the CTCs dataset to establish a more suitable model inthe later testingtumorsCirculating tumor cellis an important marker for earlyscreening and prognosis ofIn addition CTCsoriginating from the primary tumor may be more eï¬ectivefor tumor tissue tracing and molecular classiï¬cation Imagerecognition can only obtain the characteristics ofthe cellsurface If strict tissue tracing is required other molecularbiological experimental data such as the isolation of CTCcells and single cell sequencing may be required Besidesin this study we also evaluated the performance of AlexNetmodel in variant types of cancers Supplementary Table S2and Figure S2 showed that our model presents a betterperformance in Lung cancer than Gastrointestinal cancer andBreast cancer One of the reasons may be that the trainingdata size of Lung cancer is much larger than those ofGastrointestinal cancer and Breast cancer Furtherpostoperative recurrence may occur in approximately of patients even after complete resection of NSCLC Yano These proteins especially epithelial proteinssuch as EpCAM PIK3CA AKT2 TWIST and ALDH1may have more activitiesHanssen whichwilllead more inï¬uence in the morphology of cells andaï¬ecting the recognition performance thereby Therefore themultiimage omicsincluding CT images HE staining andimmunohistochemical images as well as the sequencing datamay be urgently needed at this stageFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCONCLUSIONIn orderIn the present study we established a CTC cell recognitionsoftware based on deep learningto make itmore practical we collected samples from the real worldinstead of using the public databases We performed theCTC enrichment and imFISH experiments and screened theï¬uorescence images according to the ï¬gures quality In order toimprove the eï¬ciency we used the machine instead of ngmanual screening First the pythons package was used to mage segmentation The obtained recognition sensitivity andspeciï¬city are and respectively In addition therecognition sensitivity and speciï¬city can also reach to and respectively using CNN instead of manual interventionIn the future studies we willfocus on the improvementof the accuracy and sensitivity with a more suitable deeplearning model promoting this technology to the clinic assoon as possibleDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorCTCs Recognition by Machine Learninglegal guardiannextstudy was provided by the participantsof kin Written informed consent was obtained from theindividuals and minors legal guardiannext of kin for thepublication of any potentially identiï¬able images or data includedin this AUTHOR CONTRIBUTIONSGT YL BH and QZ conceived the concept of the work BH QLJL PB HY and SL performed the experiments QL and BH wrotethe manuscript CP and HY reviewed the manuscript All authorsapproved the ï¬nal version of this manuscriptFUNDINGThis research was funded by Hunan Provincial Innovation2018RS3105Platform and Talents Program NotheNaturalNo Science Foundation of Chinathe Natural Science Foundation of Hunan Province No2018JJ3570 and the Project of Scientiï¬c Research Fundof Hunan Provincial Education Department Nos 19A060and 19C0185ETHICS STATEMENTSUPPLEMENTARY MATERIALThe studies involving human participants were reviewed andapproved by The Ethics Committee of Chifeng MunicipalHospital Written informed consentto participate in thisThe Supplementary Materialonline202000897fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389fbioeatREFERENCESAnand K and Roszik J Pilot study of circulating tumor cells in earlystage and metastatic uveal melanoma Cancers Basel 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Peeters D J The clinical use of circulating tumor cells CTCs enumerationfor staging of metastatic breast cancer MBC International expert consensuspaper Crit Rev Oncol Hematol 101016jcritrevonc201812Dominguez C Heras J and Pascual V IJ CV combining ImageJand CV for processing images in biomedicine Comput Biol Med 101016jcompbiomed201703027Dong J Zhu D Tang X Qiu X Lu D Li B Detectionof circulating tumor cell molecular subtype in pulmonary vein predictingprognosis of stage IIII nonsmall cell lung cancer patients Front Oncol 103389fonc201901139Erickson B J Korï¬atis P Akkus Z and Kline T L Machinelearning for medical imaging Radiographics 101148rg20171Ferreira M M Ramani V C and Jeï¬rey S S Circulating tumortechnologies Mol Oncol 101016jmolonc2016cellGabriel M T Calleja L R Chalopin A Ory B and Heymann D Circulating tumor cells a review of nonEpCAMbased approaches for cellenrichment and isolation Clin Chem 101373clinchem2015Grover P K Cummins A G Price T J RobertsThomson I C andHardingham 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clinicalrelevance of EpCAM Cell Stress 1015698cst2019and Pantel K BiologySKloten V Lampignano R Krahn T and Schlange T Circulatingtumor Cell PDL1 expression as biomarker for therapeutic eï¬cacy ofimmune checkpoint inhibition in NSCLC Cells 103390cells808Koudelakova V Trojanec R Vrbkova J Donevska S Bouchalova K KolarZ Frequency of chromosome polysomy in relation to CEP17copy number in a large breast cancer cohort Genes Chromosomes Cancer 101002gcc22337Kraeft S K Ladanyi A Galiger K Herlitz A Sher A C Bergsrud D E Reliable and sensitive identiï¬cation of occult tumor cells usingthe improved rare event imaging system Clin Cancer Res 10115810780432ccr030361Krizhevsky A Sutskever I and Hinton G ImageNet classiï¬cation withdeep convolutional neural networks in Paper Presented at the NIPS LakeTahoe Harrahs and HarveysLe N Q Ho Q T and Ou Y Y Incorporating deep learning withconvolutional neural networks and position speciï¬c scoring matrices foridentifying electron transport proteins J Comput 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previous studies have shown a strong coexistence of colorectal neoplasia crn and cardiovascular diseases cvd this study was aimed to summarize the available evidence on association of cvd risk with early crn detection in asymptomatic populations pubmed web of science and embase were systematically searched for eligible studies published until dec studies exploring the associations of recommended cvd risk assessment methods eg risk scores carotid artery plaque and coronary artery calcium score [cacs] with risk of crn were included metaanalyses were conducted to determine the overall association of cvd risk with the crn a total of studies were finally included the association of carotid artery plaque with the risk of colorectal adenoma ad was weakest pooled odds ratio [or] confidence interval [ci ] participants with cacs100 had about 2fold increased risk of ad than those with cacs0 the pooled ors were ci and ci for the risk of advanced colorectal neoplasia an and ad respectively in participants with framingham risk score frs20 when compared to participants at low risk frs10 frs might help identify subgroups at increased risk for an but further studies are needed keywords cardiovascular disease risk assessment colorectal neoplasiaintroductionboth colorectal cancer crc and cardiovascular diseases cvd are the leading causes of mortality and morbidity worldwide12 previous studies have shown a strong coexistence of colorectal neoplasia crn and cvd probably due to the shared risk factors eg smoking obesity and metabolic syndrome and pathophysiological mechanisms eg chronic inflammation and oxidative stress3current guidelines8 recommend assessing the cvd risk in healthy people using risk estimation scores such as framingham risk score frs1112 procam13 and the pooled cohort equation14 which are based on individuals medical history and easily available laboratory data in addition assessment of subclinical atherosclerosis by imaging modalities could be added as risk modifiers to help make clinical decisions for borderline or intermediaterisk adults8 routine use of imaging modalities is not recommended for cvd risk assessment in clinical practice due to the medical costs or invasiveness but incorporation of imaging data such as the anklebrachial index abi coronary artery calcium score cacs and carotid artery plaques cap could improve the prediction of cvd risk15clinical epidemiology chen this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cchen dovepressvarious risk scores have also been developed for predicting advanced colorectal neoplasia an18 although several studies2526 have reported that elevated blood lipids the well documented cvd risk factor and history of cvd were associated with increased risk of crc the majority of risk scores developed for an did not include them into the models27 recent studies have reported the associations between cvd risk assessment and risk of1 crn higher frs estimating the 10year risk of developing coronary heart disease chd1112 was significantly associated with the higher risk of an frs vs frs10 odds ratio [or] confidence interval [ci] abi was associated with 13fold increased risk of an in a recent study29 cap and cacs were also found to be positively related to the increased risk of adenoma ad and an in several studies30given a number of shared risk factors and mechanisms between cvd and crc and the emerging epidemiological evidence of association between cvd risk and crc there is a possibility that cvd risk assessment could help trigger crc screening therefore the aim of this review was to provide an overview of the cvd risk assessment methods and their associations with the risk of crn fully understanding of the current knowledge and existing gap might promote better prevention and treatment for cvd and crc circulating and urinary biomarkers have either no or only limited value when added to cvd risk estimation score systems834 thus only score models and imaging methods recommended as risk modifiers abi cacs and cap in the guidelines8 were included in this reviewmaterials and methodsthis systematic review was conducted following the procedure recommended by the cochrane collaboration35 and was reported according to the preferred reporting items for systematic reviews prisma checklist36 ethical approval and patient informed consent were not necessary since all the data included in the current study were obtained from previously published studiesand metaanalyses remaining publications and reference lists were scrutinized studies that fulfilled the predefined criteria were includedinclusion and exclusion criteriawe required that included studies meet the following criteria published as an original research in a peer reviewed cardiovascular risk has been assessed using either score models or imaging methods recommended as risk modifiers abi cacs and cap in the guidelines3 only included participants who were considered asymptomatic reported the association of cvd risk assessment results with the risk of crn studies were excluded if they were published as conference proceedings dissertations or s only or were not published in english pico eligibility criteria for this review were presented in the supplementary table s1data extractiontwo authors yc and xc independently performed data extraction of all included studies the following information was ed author publication year study period number of participants age number of males outcome ad an and so on data source medical records questionnaires or both cvd risk assessment and association indexdiscriminatory accuracy or hazard ratio [hr] specificity sensitivity or area under the receiver operator characteristic curve values] in case of any disagreement consensus was obtained by discussionquality assessment in eligible studiesrisk of bias and applicability were assessed according to quality assessment of diagnostic accuracy studies2 quadas237 quadas2 evaluates the risk level of bias composed of four basic components patient selection index test reference standard flow and timing clinical applicability is also assessed for the first three components the risk of bias and concerns regarding applicability for each study was then rated as high low or unclearliterature search strategiespubmed embase and web of science were searched up to december to identify the relevant papers the searched items were presented in the appendix which mainly covers expressions for cvd risk score models recommended imaging modalities crn and discriminatory accuracy or strength of association after removal of duplicates titles and s of records were screened according to the inclusion and exclusion criteria full texts of the statistical analysiswe pooled ors for the same cvd risk assessment index using r statistical software version and the r meta package version for frs and cacs ors were pooled separately for different levels of scores using the lowest level as reference two kinds of outcomes ad and an were reported in the studies using frs for cvd risk assessment and thus ors were pooled separately for different outcomes heterogeneity across studies was evaluated submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen using cochranes q statistic with p value and the i2 statistic if significant heterogeneity was observed i2 or pqstatistics a randomeffects model was used to calculate pooled estimates otherwise a fixedeffects model was used35 twosided p values of or lower were considered to be statistically significantresultsliterature search resultsa total of records were obtained in the initial search including citations from pubmed citations from embase and citations from web of science after removal of duplicates n1609 and exclusion due to our predefined criteria n5727 records were qualified for fulltext assessment fortyfour records were excluded due to the inclusion and exclusion criteria finally a total of studies28 including one study which was identified through crossreferences were included the detailed information of the selection process was presented in figure study characteristicstable summarized the basic characteristics of the included studies published between and of the included studies nine were from korea and the other three studies were from japan austria and turkey respectively the study periods stretched from to with sample sizes ranging from to only one was designed as a prospective study41 and the others were crosssectional studies most studies included participants aged older than years and only one study enrolled subjects aged years32 in addition most studies were predominantly in men with proportions of males among participants ranging from to four cvd risk assessment methods abi cap cacs and frs were used in the included studies all studies explored the role of cvd risk assessment method on the detection of ad and some of risk adenoma3032 and an2829384243focused on colorectal high them also figure flowchart of inclusions of studies about relation of cvd risk to crn note adapted from moher d liberati a tetzlaff j preferred reporting items for systematic reviews and metaanalyses the prisma statement plos med creative commons license and disclaimer available from httpcreativecommonslicensesby40legalcode36 abbreviations cvd cardiovascular disease crn colorectal neoplasiaclinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable basic characteristics of included studies about relation of cvd risk to colorectal neoplasiastudycountrystudy periodnumber of participantsyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj 2019a41lee jy niederseer d basyigit s japankoreakoreakoreakoreakoreakoreakoreakoreakoreaaustriaturkeyage years mean±sd ± ± 530b median± ± ± ± 526b± ± ±male n outcomec data sourcececum intubation ratedcvd risk assessment ad anad hraadad anad hraadadadadad anad anad anmrqmrqmrqmrmrqmrqmrmrmrqmrqmrqmrqnrnrnrnr¥nrabicapcapcapcacscacscacscacscacsfrsfrsfrsnotes ait is a retrospective followup study and all the other studies are crosssectional bsd was not reported cdetected by colonoscopies in all included studies d100 cecum intubation rate participants with failure of cecum intubation were excluded nr not reported studies mentioned that colonoscopies were extended to cecum in the methods section but did not reported the success rate of cecum intubation abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score hra high risk adenoma mr medical records nr not reported q questionnaires sd standard deviationquality assessment of studiesthe results for the quality of included studies using the quadas2 tool are presented in table regarding patient selection one study by kim did not provide detailed information about patient selection31 thus the risk of bias and applicability concerns were rated unclear for this domain in this study otherwise no major risk of bias or applicability concerns were identifiedassociation of cvd risk assessed by different methods with crc risktable described the details of the cvd risk assessment methods in the included studies abi was associated with 13fold ci increased risk of an29 three studies reported the weak association between cap and risk of ad303138 one of them also showed an increased risk of an in the participants with cap but the results were not statistically significant or table risk of bias and applicability judgements in quadas2studyrisk of biasapplicability concernstotalpatient selectionindex testreference standardflow and timingpatient selectionindex testreference standardyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj lee jy niederseer d basyigit s totalnotes _ high risk low risk unclear risksubmit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen table details of the cvd risk assessment methods in the included studies about relation of cvd risk to colorectal neoplasiastudycategoriesboutcome or[ ci]yamaji y kim j abnormal abiabnormal abicap yescap yeskim h cap yescha jm yun ke choi sh cap yescap yescacs cacs cacs cacs cacs cacs cacs cacs cacs yang mh201339cacs kim hb cacs cacs cacs ¥lee yj 2019a41cacs lee jy niederseer d basyigit s frs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highadanadhraadadanadadadhrahrahraadadadadadadadadadadananadadananadadanan[ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]hr [ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]notes ain participants without adenoma cacs0 at baseline compared to cacs0 increased the risk of colorectal adenoma at followup colonoscopy hr ci bthe lowest level was defined as reference abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque frs framingham risk score hra high risk adenoma hr hazard ratio or odds ratio ci confidence interval ci in addition the presence of cap was associated with increased risk of colorectal high risk adenoma or ci four studies reported ors for different levels of cacs with cacs0 as reference32333940 highest cacs levels seemed to be associated with the increased risk of ad with or ranging from to the 10year chd risk estimated by frs was categorized as low risk intermediate risk and high risk ¥ participants with high risk of 10year chd had increased risk of either ad or an in the study by basyigit participants at high chd risk had about 4fold or ci increased risk of an28metaanalyses of available ors for different cvd risk assessment methodsmetaanalyses were performed in the studies that provided ors and their cis for the same cvd risk assessment index the association of cap with the risk of ad was weakest the pooled or ci a medium level of cacs cacs was associated with 134fold increased risk of ad when compared to the lowest category of cacs cacs0 participants with cacs100 had an increased risk of ad and the pooled or was ci the pooled ors were ci and ci for the risk of an and ad respectively in participants with high chd risk frs20 when compared to participants at low chd risk frs10 further details were presented in table and in the supplementary figures s1discussionthis systematic review summarized the associations of recommended cvd risk assessment methods with risk of crn in asymptomatic populations a total of studies including four different methods were identified among these methods frs was most strongly associated with risk of both an and ad participants with frs20 have about 34fold and 23fold increased risk of an and ad respectively when compared to participants at low chd risk frs10 only one study29 reported that abnormal abi greatly increased the risk of an thus it was not included in the metaanalysisboth crc and cvd are thought to develop via a process of insulin resistance inflammation and oxidative clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable metaanalysis of odds ratios for different cvd risk assessment toolsstudycvd risk assessmentcategoriesaoutcomeor cicapcacscacscacsfrsfrsfrsfrsyes vs nocacs vs cacs0cacs vs cacs0cacs vs cacs0intermediate vs low riskhigh vs low riskintermediate vs low riskhigh vs low riskadadadadadadanan note athe lowest level was defined as reference abbreviations ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score or odds ratio ci confidence intervalstress74547 which might partially explain why they share a number of risk factors eg alcohol consumption tobacco use physical activity use of antiinflammatory agents obesity and diabetes mellitus4548 in addition several cellular metabolismrelated pathways eg ampk and pparγ signaling pathways eg wnt signaling pathway and genetic pathways eg lrp6 mutation and tcf7l2 polymorphism are not only associated with accelerated atherosclerosis and an increased risk of cvd but also linked to cancer development and progression7 better understanding of these overlaps might promote shared management of prevention and treatment for both disordersrisk of an in this review the strength of associations between identified cvd risk assessment methods and the risk of crn was generally weak except frs which was modestly associated with frs20 vs frs10 frs was calculated based on age total cholesterol highdensity lipoprotein cholesterol smoking status systolic blood pressure and treatment of blood pressure which are typically available in the medical records44 compared to the more sophisticated risk calculators232449 for predicting an which need variables such as physical activity red meat intake and vegetable consumption frs has relatively higher generalizability and lower recall bias a recent study has recommended the combined preventive screening and research efforts in the prevention of both cvd and cancer50 if participants with highrisk of cvd predicted by frs could be recommended to have a screening for crn which will help increase compliance and uptake of crc screening as persons who are aware of their increased risk are more likely recommendations furthermore it also maximizes the medical values of the comply with to expert information participants obtain from a clinical examination or risk assessment and thus reduces the time and costs for health carehowever there are some issues that merit our attention firstly the included studies are all crosssectional which limits the comparisons between frs and the previously developed risk prediction models for crc secondly frs has its own limitations frs only estimates 10year chd risk for all individuals years or older but not the overall cvd risk in addition it is developed based on the american population while most of study participants are asians in the included studies studies have shown that frs overestimated cvd risk in the asian cohorts51 at last the included studies tended to yield results with wide ci probably due to the limited number of participants the wider the ci the less the precision in summary higher cvd risk might trigger concurrent crc screening which should be further validated on largescale studies and future studies could consider about using the overall cvd risk score models developed from data of local cohorts to predict the risk of crcas for imaging data the association of cap or cacs with risk of ad is not strong enough that imaging index alone might not be useful for informing early detection of crn similarly routine screening with imaging modalities to predict future cardiovascular events is generally not recommended in clinical practice but use of these imaging techniques has been shown to improve cvd risk assessment and serve as a guide for initiating preventive therapies8 a high cacs can help modify the predicted risk obtained from frs alone especially among patients in the intermediaterisk category16 up to now only one risk score developed in the multiethnic study of atherosclerosis mesa study used both cacs and submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen traditional risk factors to predict the 10year chd risk55 inclusion of cacs in the mesa risk score offered significant improvements in risk prediction cstatistic vs p factors in the risk models like smoking behaviors and blood lipids are closely related to the incidence and progression of cvd but they are not direct markers of current status of atherosclerosis this might help explain why the performance of risk models is improved by adding markers with anatomical delineation through imaging technology accounting for the higher performance of the combined use of risk scores and imaging tools on cvd risk assessment further studies could consider about exploring the association of combined form of them with the risk of crcwe also observed that less than half of included studies reported the associations of cvd risk with both risk of an and ad2829384243 colonoscopy is considered to as a valid primary screening tool for crc and is able to detect both ad and an the lower prevalence of an and the limited number of participants in several included studies might limit the power to explore the relation of an with cvd risk which could partly explain why most of studies did not include an as outcome therefore the findings should be carefully interpreted and further validated on largescale studiesour study has some strengths comprehensive search strategies along with welldefined eligibility criteria were used to help identify relevant s in addition two reviewers independently extracted data and assessed the risk of bias in the included studies however several limitations should also be addressed firstly the current meta analysis was based on observational studies there were the possibilities of potential effects of unknown or residual confounding factors on our results secondly as we only considered about established cvd risk models and recommended imaging modalities the potential of other cvd risk assessment index on the detection of crn was not summarized and compared in this study however it is also reasonable to just include these methods since their feasibility and performance for cvd risk prediction have been well approved in the clinical practice thirdly cut off values and group comparisons for the same cvd risk assessment method varied in the included studies which limits the synthesis of results for example the cut off values for cacs are the tertiles of cacs in the study by kim 40 however cacs was categorized into three groups with cut off values at and in the other studies3233 therefore less studies were included in the metaanalysis which might influence the accuracy of the pooled results lastly most of studies were conducted in asian populations which is an inherent limitation of the included studies thus our findings might not be applicable to other populations and needs to be externally validated in racially diverse populationsconclusionsto our knowledge this is the first review that applies metaanalyses to determining the overall association of recommended cv risk assessment methods with the risk of crn in the asymptomatic population frs calculated based on shared risk factors of cvd and crc shows potential to help identify subgroups at increased risk for an whether the combination of frs and imaging index is useful for the optimal evaluation of crn risk remains to be solved in the future studies cvd risk might inform crc screening which needs more research in the future to validate its feasibility and effectivenessabbreviationsabi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque chd coronary heart disease ci confidence interval crc colorectal cancer crn colorectal neoplasia cvd cardiovascular disease frs framingham risk score hr hazard ratio hra high risk adenoma mr medical records nr not reported or odds ratio prisma preferred reporting items for systematic reviews and metaanalyses quadas2 quality assessment of diagnostic accuracy studies2 q questionnaires sd standard deviationfundingthis research was funded by national natural science foundation of china grant number disclosurethe authors report no conflicts of interest in this workreferences bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin doidoi103322caac21492 joseph p leong d mckee m et al reducing the global burden of cardiovascular disease part the epidemiology and risk factors circ res doidoi101161circresaha117308903 chan aoo man hj kwok fl et al prevalence of colorectal neoplasm among patients with newly diagnosed coronary artery disease j am med assoc doidoi101001jama29812 clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepress chan aoo lam kf tong t coexistence between colorectal canceradenoma and coronary artery disease results from patients aliment pharmacol ther doi doi101111j13652036200602958x wang sc schulmanmarcus j fantauzzi j et al colon cancer laterality is associated with 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" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus piv respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecaloral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled toilet plume in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as the quintessential perpetrator of inflammation to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1 generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a gray zone about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a one size fits all consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should never worry about action only inaction 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o | 0 |
Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these ï¬ndings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insuï¬cient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social ï¬nancialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreï¬ect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Speciï¬cally procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and speciï¬city[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical eï¬cacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBG level ¥ mmolling mgdl or h blood glucose ¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushings syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coeï¬cients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Students ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe MannWhitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r P There was a significant negative correlationbetween OC or CTX and TG rs P rs P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs P OC rs P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Theseï¬ndings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars P lmgp HTPlmgn XTCð½r P HbA1c brs P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearsons correlation coeï¬cient rs Spearmans correlation coeï¬cient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coeï¬cients Standardized coeï¬cients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These ï¬ndings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen ï¬bres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and conï¬rmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliï¬ozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliï¬ozin dapagliï¬ozin and empagliï¬ozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third theuence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespeciï¬c alkalinephosphatase BAP vitamin D or steroids all of which uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its speciï¬city for bone metabolism is lacking [] BAPis a more bonespeciï¬c marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high speciï¬city in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreï¬ects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These ï¬ndings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DMuencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneï¬cial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the ï¬ndings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conï¬ict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientiï¬c Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang IDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and Diabetes Research and Clinical Practicevol pp [] A V Schwartz D E Sellmeyer K E Ensrud Olderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp [] L Forsen H E Meyer K Midthjell and T H Edna Diabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp [] Y Fan F Wei Y Lang and Y Liu Diabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp [] M Janghorbani R M Van Dam W C Willett and F B HuSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides Vitamin D and glycemic control in diabetes mellitus type Therapeutic Advances in Endocrinologyand Metabolism vol no pp [] H Miyake I Kanazawa and T Sugimoto Association ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalciï¬ed Tissue International vol no pp [] E S Siris R Adler J Bilezikian The clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp [] M B Greenblatt J N Tsai and M N Wein Bone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell Markers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp [] H W S Cabral B F G Andolphi B V C Ferreira Theuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp [] P Iglesias F Arrieta M Pi±era Serum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp [] J M Wishart A O Need M Horowitz H A Morris andB E C Nordin Eï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp [] P Szulc P Garnero F Munoz F Marchand and P D Delmas Crosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp [] K G M M Alberti P Z Zimmet and WHO ConsultationDeï¬nition diagnosis and classiï¬cation of diabetes mellitusand its complications Part diagnosis and classiï¬cation ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp [] J StarupLinde and P Vestergaard Biochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp [] L Achemlal S Tellal F Rkiouak Bone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin Association of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira Metabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco Bone turnover markers in men andwomen with impaired fasting glucose and diabetes Calciï¬edTissue International vol no pp [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann Lower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu Signiï¬cant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp [] A Tan Y Gao X Yang Low serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang The relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp [] Y Chen Q Zhao G Du and Y Xu Association betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a | 2 |
" atopic dermatitis ad is a worldwide chronic skin disease which burden public health sea buckthornsbt hippophae rhamnoides l elaeagnaceae oil as a traditional herbal medicine has been used for diseasetreatment for many years the effects of sbt oil on ad mouse model induced by repeated administration of dinitrochlorobenzene dncb in balbc mice was evaluated in this studymethods mice were divided into four groups including the normal control group ad model group ad modelgroup treated with sbt oil mlkg and ad model group treated with sbt oil mlkg same volume at differentconcentrations of sbt oil was applied daily on the latter two groups by gavage for days following ad modelinduction the function of skin barrier and the production of il4 ifnÎ tnfα and tslp were examined afteranimal sacrifice the migration and mature of langerhans cell lcs in lymph node was further assessed by flowcytometryresults sbt oil alleviated dermatitis scores decreased ear thickness prevented infiltration of mast cell reducedlymph node weight and depressed activity of th2 cells sbt oil also reduced the expression of il4 ifnÎ tnfα andtslp in ear tissue ige level in serum and mrna relative expression of il4 ifnÎ tnfα in lymph node moreoversbt oil inhibited the migration of lcs cells from local lesions to lymph node and its mature in lymph nodes these results suggest sbt oil had a beneficial effect either systemic or regional on dncbinduced admice via maintain the balance of th1th2 and may be a potential complementary candidate for ad treatmentkeywords atopic dermatitis sea buckthorn oil 24dinitrochlorobenzene cytokine ad is a chronic inflammatory skin disease characterizedwith eczematous pruritic rash which has high morbidityin children and could be recurrent in adulthood [ ]as a general public health disease the prevalence of adhas increased in recent years [ ] ad affects nearly correspondence hongquanguansinacom houdiandong163com xinxin wang and sijia li contributed equally to this work5college of integrated traditional chinese and western medicine liaoninguniversity of traditional chinese medicine chongshan road no79shenyang liaoning pr chinafull list of author information is available at the end of the of children and of adults worldwide and the incidents become higher and higher although thepathogenesis of ad is not explicit utterly genetic riskenvironmental factors skin barrier dysfunction and immune dysregulation are thought to play important rolesduring the pathogenesis of ad [] as for immunedysregulation th2 skewing seems to be the key point ofad pathogenesis [ ] immunological disorder ofth1th2 balance due to strong type immune responses characterized by over infiltration of mast cellincreased production of th2 cytokines and ige level in the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc complementary medicine and therapies page of serum plays crucial role in the onset and process of adthese th2 cytokines subsequently induce the release ofother proinflammatory cytokines such as ifnÎ throughactivating of th1 cells in clinical practicedue to the heavy burden ad placed on society and patients [ ] treatment approaches are needed imperativelyregional emollient andsystemic corticosteroids were generally used to cure ad[ ] however experts ofinternational eczemacouncil reached on a that although the useof corticosteroids for ad is widespread it is also discouraged due to the side effects and the risk of reboundin consideration of potential fearful side effect of topicalsteroid and immunosuppressive application [ ]there is a strong enthusiasm in seeking alternative andcomplementary medication to treat ad recently seeking new potential candidate from natural materials forad management attracted greatly attention [ ]sbt is a wild deciduous shrub or dwarf tree belongingto the elaeagnaceae family which has been used in tibetan mongolian and chinese traditional medicine extensively for disease management [] according tomany researchers sbt has various medicinal effects suchas antitumor antistress antiinflammatory antiulcerantioxidant healing regulation of cardiovascular andimmune system [] sbt oil which contains richfatty acids tocopherols Ï3 and Ï6 etc is a main bioactive part of sbt and it has been proved to have beneficial effect on skin inflammation conditions and have theability to improve the composition of fatty acid in skin[ ] therefore this study carried out to explore thebeneficial effects of sbt oil on dncbinduced admouse model and its possible mechanismmethodssbt oilsbt oil was provided by liaoning dongning pharmceutical co ltd the oil was extracted from thedried press residue including berry flesh seeds andpeel of sbt juice processing by aseptic supercriticalcarbon dioxide process analysis of samples was performed using a hp5ms capillary column m mm μm agilentinc santaclara ca usa in a gcms 5975c agilent technologies inc sample was injected into the columnand ran using split mode split ratio the helium carrier gas was programmed to maintain atechnologiesconstantflow rate of mlmin oven was initially °c for min then finally raised to °c at °cmin fatty acids were identified by a reference standard mixture fame supelco bellefonte pa usa analyzed under the same operating conditions as thoseemployed for fame of the samples the componentsin sbt oil are exhibited in table animals and animal treatmentfemale healthy specific pathogenfree balbc miceaged weeks weighted ± g were provided byliaoning changsheng biotechnology co ltd benxichina all mice were housed in groups of mice percage waiting to be grouped in a specific pathogenfreeenvironment in h lightdark cycle and allowed free towater and food mice were acclimatized for week before ad model induction mice were randomly dividedinto groups the normal control group ad modelgroup ad model group treated with sbt oil mlkgand ad model group treated with sbt oil mlkgeach group with mice the dorsal skin of each mousewas shaved following dncb μl1 sensitizationthree times in total from day to day and the skin ofleft ear was challenged by dncb μl05 seventimes in total from day to day ad model grouptreated with sbt oil mlkg was given ml sbt oilplus ml olive oil per mice ad model group treatedwith sbt oil mlkg was given ml sbt oil permice oil was applied by intragastric administration oncea day from day15 to day olive oil ml was givenfor the normal control group and ad model group atthe same time at the same time the thickness of left earwas measured every days all animals were sacrificedat day and samples including blood left ear tissuesand submaxillary lymph nodes were collected the micewere anesthetized with avertin solution g tribromoethanol powder dissolved into ml 2methyl2butanol and ml pbs at °c which was filtered usinga nalgene μm filter thermo fisher scientific incwaltham ma usa and sacrificed via exsanguination the fullscale procedures of ad model inductionand treatment are shown in fig all experimental procedures performed were approved by the ethical committee of experimental animal careat liaoninguniversity of traditional chinese medicine shenyangpr chinatable major fatty acids and contents of sitosterol and βcarotene in sbt oilfatty acids myristic acidc140palmitic acidc160palmitoleicacidc161stearic acidc180oleic acidc181linoleic acidc182linolenic acidc183sitosterolmggβcarotenemgg 0cwang bmc complementary medicine and therapies page of fig general schematic diagram for ad model induction and sbt oil treatmentevaluation of ad severitydermatitis severity was assessed by ear thickness anddermatitis scores through the method of blind ear thickness was measured every days since day with a digitalthickness gauge mitutoyo co kanagawa japan dermatitis scores was calculated according to main characteristics drynesscrusting hemorrhageerythema erosionexcoriation and edema each one was marked on ascale from none mild moderate to severethe overall dermatitis score was consist of the sum of individual scores which range from to μm thick werehistopathological analysisthe left ear samples of mice were collected on day then fixed in formalin and embeded in paraffin thesectionseither withhematoxylin and eosin he for visualizing dermal andepidermal thickness or with toluidine blue tb for visualizing mast cell numbers the mast cells were countedin sections of power fields at magnificationstainedimmunohistochemistryin short after deparaffinization and rehydration the eartissue slides were treated with hydrogen peroxidemethanol for inhibiting endogenous peroxidase and withhigh pressure for antigen retrieval then the slides wereincubated with sheep serum for min at °c withprimary antibodies abcam for overnight at °c withsecondary antibodies provided by zhongshanjinqiaobeijing china for h at °c at last the slides werestained with diaminobenzidine dab provided byzhongshanjinqiao beijing china for coloration resultwas analyzed by imagejrealtime polymerase chain reaction rtpcrmouse submaxillary lymph nodes were collected andweighted while sacrifice and total rna was extractedfrom lymph node tissues using trizol reagent invitrogen thermo fisher scientificfollowing theincmanufacturers protocols and reverse transcribed withprime scripttmrt reagent kit takara biotechnologyco ltd dalian china realtime polymerase chain reaction analyses were performed under the protocols ofsybr®premix ex taqtm ii takara biotech co ltddalian china and the primers used in this study weredesigned as shown in table relative quantities of alltargets in test samples were normalized to their corresponding gapdh levels the 2δδct method was usedto calculate relative expression quantifyflow cytometrythe antibodies used for flow cytometry were providedby bd usa and the scheme was performed follow induction about cells of submaxillarylymph node was collected in ep tube centrifuged at rpm and °c for min one hundred μl pbs wasmixed with cells after discarding the supernatant thenanother μl pbs containing fluorescent antibodieswas added for staining at °c for min and the processwas kept out of the sun five hundred μl pbs was addedand blended repeatedly for washing then centrifuged at rpm and °c for min the supernatant was discarded carefully and another μl pbs was added andmixed finally the mixture was transferred to flow tubefor flow cytometrytable primers used for rtpcrgeneil4ifnÎtnfαgapdhprimer sequenceforward ²acaggagaagggacgccat3²reverse ²gaagccctacagacgagctca3²forward ² tgagctcattgaatgcttgg ²reverse ² ggccatcagcaacaacataa ²forward ²ggaaaggacggactggtgta3²reverse ² tgccactggtctgtaatcca ²forward ²tggtgaaggtcggtgtgaac3²reverse ²actgtgccgttgaatttgcc3² 0cwang bmc complementary medicine and therapies page of statistical analysisthe data is presented as mean ± sd the significance ofdifferences of different groups was evaluated by oneway analysis of variance anova followed by the dunnett t test p005 was considered statistically significantresultssbt oil has a beneficial effect on skin against thedevelopment of dncbinduced ad models in balbcmiceto investigate the effect of sbt oil on adlike skinlesions in our model sbt oil mlkg10 mlkg wasapplied by gastric administration once a day followingthe ad model induction by dncb showed in fig topical application of dncb including sensitizationand challenge induced adlike skin lesions presenting as erythemaitching and hemorrhage companiedby abnormal scratching marks and dryness dermatitisseverity was assessed by ear thickness and dermatitisscores ear thickness was measured every days sinceday and the dermatitis scores was evaluated according to main characteristics as described previously after sbt oil administration for days theear thickness and dermatitis scores were significantlydecreased in a dosedependent manner compared tothe ad model group fig fig effects of sbt oil on ad model skin induced by dncb a examples of characteristic of adlike skin lesions b the ear thickness of the micec the dermatitis scores were summarized by the sum of scores according to various ad symtoms p p 005vs the control group p p vs the ad model group 0cwang bmc complementary medicine and therapies page of sbt oil contributed to the skin barrier repair anddecreased infiltration of mast cell in ad model miceinduced by dncbto evaluate the effect of sbt oil on adlike skin lesions histologically he and tb staining were performed on tissue slides repetitive application ofdncb induced dermal thickening epidermal hyperplasia and increased mast cellinfiltration in admodel group while according to he staining slidesthe dermal and epidermalthickness were both decreased and the epidermal hyperplasia was suppressedafter sbt oil administration for days which relatedto dosage fig 3a b according to tb staining slidesthe infiltration numbers of mast cell were also decreased in mice treated with sbt oil fig 3b csbt oil decreased the lymph node weights in ad modelmice induced by dncbthe submaxillary lymph nodes were collected andweighted after mice sacrifice to estimate whether sbtoil play a part in the process of ad induction the results indicated an increase in submaxillary lymph nodeweights in ad model group which was decreased bysbt oil administration fig sbt oil inhibited the expression of il4 ifnÎ tnfα andtslp in ear tissue in ad model mice induced by dncbin order to evaluate the effects of sbt oil on regulationof th1th2 cytokines the expression of il4 ifnÎtnfα and tslp in ad model mice was examined byimmunohistochemistry staining on ear tissue slides results demonstrated an upregulation expression of il4fig effects of sbt oil on histological ear skin tissue a he staining thickness in hestained tissue mast cell number in tb stained tissue c tb staining epidermis mast cell dermis b dermal and epidermal 0cwang bmc complementary medicine and therapies page of fig weights of submaxillary lymph node a submaxillary lymph node b lymph node weight a normal control group b ad model groupc treated with sbt oil mlkg d treated with sbt oil mlkg p p vs the control group p p vs the admodel groupifnÎ tnfα and tslp in ad model group which wasinhibited dosedependent by application of sbt oilfig sbt oil downregulated ige level in serum and mrnarelative expression of il4 ifnÎ and tnfα in lymphnodewe next measured ige levelin serum by elisa andmrna relative expression of il4 ifnÎ and tnfα inlymph node by rtpcr we found that ige levelinserum was increased in ad model mice induced bydncb the increase was suppressed significantly ingroups treated with sbt oil in a dosedependent manner the same as above mrna relative expression ofil4 ifnÎ and tnfα in lymph node was increased inad model group and decreased in groups treated withsbt oil fig sbt oil decreased numbers of lcs in draining lymph nodeand the expressions of cd86 ox40l and mhcii on lcsin order to assess the effect of sbt oil on the maturityand migration of lcs cell in submaxillary lymph nodewe detected cell numbers expressing cd207cd326cd86 cd80 ox40l and mhc ii by flow cytometryresults as shown in fig indicated that the expressionsof cd207cd326 cd86 ox40l and mhciion lcs cellin submaxillary lymph node were all increased in admodel groups induced by dncb and decreased ingroups treated with sbt oildiscussionad is a skin inflammatory disease induced by haptenand mediated by t cells clinically the main characteristics of ad are erythema edema papule blisterbleb bullous reaction and even necrosis the pathological changes of ad were infiltration of inflammatorystudy weand tissuecellsedemain thisemployed dncbinduced ad model using balbcmice which has been proposed as an appropriate representative of human ad because of similar symptoms including skin erosion hemorrhage epidermalhyperplasia mast cellinfiltration and increased igelevel in serum etc sbt oil as a traditional herbal extracts have been proved diversified pharmacologicalactions such as antiinflammatory relieve the pressure protecting vascular endothelial cell and immunomodulatory effects [ ] the main constituents ofsbt oilinclude fatty acids such as myristic acidpalmitic acid palmitoleic acid oleic acid etc sitosterol and βcarotene fatty acids are crucial components of cell membranes and play important role inbiologicalfunction of cells some of the fattyacids are required for innate immune activation andpathogen defense sitosterol is the main constituent of many plants and vegetables and has the abilityto modulate the functions of macrophages and antiinflammation [ ] βcarotenealso has beenshown to suppress the cellular and tissue response toinflammation [ ] in view of the immunoregulation and antiinflammatory actions of sbt oil weassessed the antiad effects of sbt oil in vivotopical application of dncb followed schedule including sensitization and challenge induced adlikeskin lesions presenting as erythemaitching andhemorrhage companied by abnormal scratching marksand dryness the ear thickness and dermatitis scoreswere all significantly increased in ad model groupcompared to control group after sbt oil administration for daysthickness and dermatitisscores in groups treated with sbt oil were significantly decreased in a dosedependent manner compared to the ad model group which indicate thatsbt oil administration suppressed the development ofadlike skin lesionsthe ear 0cwang bmc complementary medicine and therapies page of fig effects of sbt oil on expression of il4 ifnÎ tnfα and tslp in ear tissue a immunohistochemical staining of il4 ifnÎ tnfα and tslpin ear tissue b aod analysis of il4 ifnÎ tnfα and tslp p vs the control group p vs the ad model groupad is recognized as a th2midiated allergic disease withover expression of th2 cytokines and increased serum igelevel being the antibody synthesized by plasma cellsige plays an essential role in some hypersensitivity suchas ad allergic asthma and allergic rhinitis ige has thecapability of elevating the production of th2 cytokinesth2 cytokines il4 induced immunoglobulin switching inplasma cells and resulting in upregulation of serum igelevel mast cell as one of granular leukocytes can releasemany cytokines to mediate inflammatory reaction and immune regulation these cytokines also participate inpathological manifestations of many allergic disorders including ad the infiltration of mast cell which wasactivated by ige is one of the key features of adlike skin 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on ige level in serum and mrna relative expression of il4 ifnÎ and tnfαin lymph node a ige level in serum b mrnarelative expression of il4 ifnÎ and tnfα in lymph node p p vs the control group p p vs the ad model grouplesions [ ] cytokines released from activated mastcells attract eosinophils into the skin which give rise toskin tissue damage histologically according to tb staining slides the numbers of mast cell infiltration in ear skintissue of ad model mice were increased by application ofdncb and were inhibited remarkably by sbt oil the results indicated that sbt oil has beneficial effects on suppression of skin tissue mast cell accumulation in dncbinduced ad mice our tb staining results indicated thatmast cells in skin tissue were scarce in control group whileabundance in ad model group which highly conform tothe pathological changes of ad the mast cell numberwas reduced remarkably after sbt oil administration insbt oil treated group compared with ad model groupthese results suggest that sbt oil has inhibiting effect ofmast cell infiltrationaccording to studies published the over expression ofth2 cytokines go hand in hand with tslp tslp whichcan strongly promote the differentiation of th0 cells toth2 phenotype through activation of dendritic celldcs was determined as a crucial factor in the induction of th2 skewing in ad the expression of tslphas been shown to be enhanced markedly in keratinocytes of ad lesions both in ad patients and in mousemodels il4 can in turn induce the synthesis oftslp by keratinocytes importantly the migration ofdcs to draining lymph node was triggered by tslpmore interesting th1 cytokines ifnÎ which can activate keratinocytes was found also elevated in ad ifnÎand tnfα can synergistically stimulate the release ofcytokines and chemokines in chronic stage of ad inour study sbt oiltreatment reduced the increasedserum ige level which was induced by dncb application moreover sbt oil treatment also reduced dncbinduced increases in expression of il4 ifnÎ tnfαand tslp in ear tissue and mrna relative expression ofil4 ifnÎ and tnfα in lymph node these resultssuggest that sbt oil ameliorated ad symptoms partlythrough the activity suppression of th1th2 cells according to the downregulated effects sbt oil did to thetslp expression in ear tissue we speculated that sbtoil may have the ability to suppress both the activationand migration of dcs cell in order to clarify our speculationflow cytometry was used to do further studyabout lcsdraining lymph node plays an important role in thepathogenesis of ad the weight and volume of lymphnode will increase company with strengthened functionwhen it is active we investigated the local lymph nodesthrough different means first of all the submaxillarylymph node weights of dncbinduced ad model micewere increased significantly which were markedly reducedafter intragastric application of sbt oil for days in adosedependent manner we further assessed the expressions of cd207cd326 mhc class ii cd80 cd86 onlcs and ox40l on cd4 t cells in lymph node by flowcytometry because the complex immune reaction of adwas mainly taken place in lymph node langerin cd207a type ii transmembrane protein is a ctype lection oflcs lcs are virtual mediators of immune surveillance and tolerance which resided at epidermis as dc subpopulation antigens both external and internal werecaptured by lcs and presented to th0 cells within theskin draining lymph node cd207 is the only surface antigen just restricted to lcs epithelial cell adhesionmolecule epcamcd326 a cell surface protein is highlyexpressed on lcs and appears to stimulate lcs migration since cd207cd326 as the main symbol of lcs 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on the maturity and migration of lc cell a the scatter diagram which indicate the proportion of cells in lymph nodeexpressing cd207cd326 mhc ii cd86 cd80 and ox40l b the histogram of abovementioned results p p 005vs the controlgroup p p vs the ad model groupmigrated into lymph nodethe proportion changesshowed that sbt oil suppressed the migration of lcs cellfrom skin lesion to draining lymph node after degradingproteins derive from extracellular environment were takenup by endocytosis or phagocytosis and captured by mhcclass ii molecules then result in peptideloaded mhc iiand migrate to the surface of antigen presenting cell waiting for recognition by cd4 t cells finally activate adaptive immune response the increased cell proportionof mhc class ii indicated the uptrend tendency of mature 0cwang bmc complementary medicine and therapies page of lcs in lymph node in dncb induced ad model micecompared with normal control mice while this uptrendtendency was inhibited by sbt oil application in micetreated with sbt oil constant epidermal lcs are immature normally and barely express costimulatory moleculessuch as cd80 and cd86 while upon lcsmaturation the expression of these costimulatory molecules was enhanced in this study sbt oil down regulatedthe expression of cd86 on lcs in lymph node which wasenhanced by dncb in ad model mice it suggested theeffect of sbt oil on inhibiting lcs maturationox40cd134 is a transmembrane protein of tumor necrosis factor receptor superfamily member which mainlyexpressed on activated cd4 t cells and upregulatedwithin inflammatory lesions on the antigenactivated tcells [ ] tslp can stimulate the expression of ox40ligand ox40l on lcs lcs expressing ox40l migratefrom skin lesion to local lymph node and induce the transformation of th0 cells to th2 cells on one hand the increased proportion of ox40l cells in lymph node ofdncb induced ad mice was suppressed by sbt oil administration which confirmed the effect of sbt oil on activation of cd4 t cells on the other hand sbt oilconduced to the normal function of lcs through renovating the keratinocyte and suppressing tslp release as aresultthe abnormal th2 skewing inflammation wasinhibited by sbt oil administrationall in all our results suggest that sbt oil inhibitedboth the migration of lcs to lymph node and its maturation in lymph node thereby inhibited the transformation of th0 cells to th2 cells and finally limited theoccurrence of th2 type inflammatory responsein summary our study results attested that sbt oil application suppressed dncbinduced adlike symptomsby downregulating serum ige level and the productionof cytokines and chemokines and regulated th1th2balance in addition our results also indicated that sbtoil treatment inhibited the migration of lcs to draininglymph node and its maturation taken together sbt oilhas excellent therapeutic effect on inflammatory skindiseases and might be a potential complementary candidate for ad treatment in further studiesit will beworthwhile to explore the mechanism of sbt oil and itsactive constituent in the treatment of adabbreviationsad atopic dermatitis dab diaminobenzidine dcs dendritic cell dncb dinitrochlorobenzene he hematoxylin and eosin ifnÎ interferonÎige immunoglobulin e il4 interleukin4 lcs langerhans cell mhcii majorhistocompatibility complex ii rtpcr realtime polymerase chain reactionsbt sea buckthorn tb toluidine blue th1 thelper th2 thelper tnfα tumor necrosis factorα tslp thymic stromal lymphopoietinacknowledgementsnot applicableauthors contributionsxxw carried out the experiment and drafting of the manuscript sjl and jplassisted to accomplish the experiment dnk and xwh carried out statisticalanalysis pl and mx did the interpretation work hqg and ddh revised theresearch and manuscript ddh designed the experiment and submitted themanuscript all the authors read and approved the final manuscriptfundingthis project was supported by the national natural science foundation ofchina grant no which was granted to diandong hou the resultsindicated in this manuscript were main achievements of the projectavailability of data and materialsall data and materials are contained and described within the manuscriptethics approval and consent to participateall experimental procedures were conducted according to the guidelinesprovided by the ethical committee of experimental animal care at liaoninguniversity of traditional chinese medicine shenyang pr chinaconsent for publicationnot applicablecompeting intereststhe authors state no potential conflict of interestauthor details1liaoning university of traditional chinese medicine shenyang liaoning prchina 2basic medical and forensic medicine baotou medical collegebaotou inner mongolia pr china 3neurosurgery department northernhospital of inner mongolia baotou inner mongolia pr china 4liaoningdongning pharmceutical co ltd fuxin liaoning pr china 5college ofintegrated traditional chinese and western medicine liaoning university oftraditional chinese medicine chongshan road no79 shenyang liaoning pr chinareceived december accepted june referencesklonowska j new cytokines in the pathogenesis of atopic dermatitisnew therapeutic targets int j mol sci choopani r treatment of atopic dermatitis from the perspective oftraditional persian medicine presentation of a novel therapeutic approach jevid based complementary altern med brunner pm guttmanyassky e leung dy the immunology of atopicdermatitis and its reversibility with broadspectrum and targeted therapiesj allergy clin immunol 20171394ss65leung dym new insights into atopic dermatitis j clin investig kim je molecular mechanisms of cutaneous inflammatory disorderatopic dermatitis int j mol sci kim j molecular mechanism of atopic dermatitis induction followingsensitization and challenge with 24dinitrochlorobenzene in mouse skintissue toxicol res hou dd sea buckthorn hippophae rhamnoides l oil improvesatopic dermatitislike skin lesions via inhibition of nfkappab and stat1activation skin pharmacol physiol campana r molecular aspects of allergens in atopic dermatitis curropin allergy clin immunol brandt eb sivaprasad u th2 cytokines and atopic dermatitis j clin cellimmunol de vuyst e atopic dermatitis studies through in vitro models frontmed lausanne park jg tabetri tabebuia avellanedae ethanol extract amelioratesatopic dermatitis symptoms in mice mediat inflamm 0cwang bmc complementary medicine and therapies page of liu r βsitosterol modulates macrophage polarization and attenuatesrheumatoid inflammation in mice pharm biol vollmer d west v lephart e enhancing skin health by oral administrationof natural compounds and minerals with implications to the dermalmicrobiome int j mol sci cicero afg colletti a effects of carotenoids on health are all the sameresults from clinical trials curr pharm des ito t il33 promotes mhc class ii expression in murine mast cellsimmun inflamm dis dubois a regulation of th2 responses and allergic inflammationthrough bystander activation of cd8 t lymphocytes in early life jimmunol girtsman t natural foxp3 regulatory t cells inhibit th2 polarizationbut are biased toward suppression of th17driven lung inflammation jleukoc biol wallmeyer l tslp is a direct trigger for t cell migration in filaggrindeficient skin equivalents sci rep leyvacastillo jm tslp produced by keratinocytes promotes allergensensitization through skin and thereby triggers atopic march in mice jinvest dermatol feinberg h trimeric structure of langerin j biol chem zhang x tim4 is differentially expressed in the distinct subsets ofdendritic cells in skin and skindraining lymph nodes and controls skinlangerhans cell homeostasis oncotarget kumkate s cd207 langerhans cells constitute a minor population ofskinderived antigenpresenting cells in the draining lymph node followingexposure to schistosoma mansoni int j parasitol gaiser mr cancerassociated epithelial cell adhesion moleculeepcam cd326 enables epidermal langerhans cell motility and migrationin vivo proc natl acad sci u s a 201210915e889 natarajan k the role of molecular flexibility in antigen presentationand t cell receptormediated signaling front immunol weinberg ad science gone translational the ox40 agonist storyimmunol rev kinnear g a diametric role for ox40 in the response of effectormemory cd4 t cells and regulatory t cells to alloantigen j immunolpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsshrestha s burden of atopic dermatitis in the united states analysis ofhealthcare claims data in the commercial medicare and medicaldatabases adv ther arima k burden of atopic dermatitis in japanese adults analysis ofdata from the national health and wellness survey j dermatol megna m systemic treatment of adult atopic dermatitis a reviewdermatol ther heidelb glatz m emollient use alters skin barrier and microbes in infants at riskfor developing atopic dermatitis plos one 2018132e0192443 drucker a | 0 |
"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Marys Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturers recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patients space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patients blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patients space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to DisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturers recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturers instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c" | 2 |
acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratiï¬cation in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple ï¬elds such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical signiï¬cance theseï¬ndings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression proï¬lesof aml specimens and the corresponding clinical followupdata were downloadedidentiï¬cation of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identiï¬ed between high and low score groups stratiï¬ed bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsigniï¬cance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identiï¬cation of gene coexpressionnetworksthrough highthroughput expression proï¬les ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbrieï¬y the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirnamrna and mirnalncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a proteinprotein interaction ppi networkof the degs identiï¬ed in the cerna network the interactingpairs with a conï¬dence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplanmeier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical signiï¬cance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskalwallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression proï¬les and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range years oldaccording to the fab classiï¬cation there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score was range from to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentiï¬cation of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identiï¬ed mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplanmeier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identiï¬ed and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identiï¬cation ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta ï¬nal lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aproteinprotein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been conï¬rmed tobe associated with immune microenvironment and leukemiaprogression association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and proteinprotein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi proteinprotein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayï¬nd new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplanmeier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors then we identiï¬ed diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identiï¬ed lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and speciï¬city ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of proammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic signiï¬cance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsigniï¬cance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identiï¬ed lncrnas with clinical signiï¬cance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identiï¬ed further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironmenthome of the leukemic blasts blood rev doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer doi 101002ijc yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identiï¬cation 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" patients who have undergone radical cystectomy for urinary bladder cancer are not sufficientlyphysically active and therefore may suffer complications leading to readmissions a physical rehabilitationprogramme early postoperatively might prevent or at least alleviate these potential complications and improvephysical function the main aim of the canmore trial is to evaluate the impact of a standardised and individuallyadapted exercise intervention in primary health care to improve physical function primary outcome and habitualphysical activity healthrelated quality of life fatigue psychological wellbeing and readmissions due tocomplications in patients undergoing roboticassisted radical cystectomy for urinary bladder cancermethods in total patients will be included and assigned to either intervention or control arm of the study allpatients will receive preoperative information on the importance of early mobilisation and during the hospital staythey will follow a standard protocol for enhanced mobilisation the intervention group will be given a referral to aphysiotherapist in primary health care close to their home within the third week after discharge the interventiongroup will begin weeks of biweekly exercise the exercise programme includes aerobic and strengtheningexercises the control group will receive oral and written information about a homebased exercise programmephysical function will serve as the primary outcome and will be measured using the sixminute walk test secondaryoutcomes are gait speed handgrip strength leg strength habitual physical activity healthrelated quality of lifefatigue psychological wellbeing and readmissions due to complications the measurements will be conducted atdischarge ie baseline postintervention and year after surgery to evaluate the effects of the intervention mixedor linear regression models according to the intention to treat procedure will be usedcontinued on next page correspondence andreaporserudkise1department of neurobiology care sciences and society division ofphysiotherapy karolinska institutet stockholm sweden2allied health professionals function medical unit occupational therapy andphysiotherapy karolinska university hospital stockholm swedenfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cporserud bmc cancer page of continued from previous pagediscussion this proposed randomised controlled trial has the potential to provide new knowledge withinrehabilitation after radical cystectomy for urinary bladder cancer the programme should be easy to apply to otherpatient groups undergoing abdominal surgery for cancer and has the potential to change the health care chain forthese patientstrial registration clinicaltrialsgov clinical trial registration number nct03998579 first posted june keywords abdominal surgery behaviour bladder neoplasm complications exercise physical activity primaryhealth care process evaluation the most common treatment for solid cancer tumoursis surgery often in combination with chemotherapy orradiotherapy or both minimising postoperative complications is important in health care for the individual patient and in reducing health care costs for society earlymobilisation at the ward and physical activity at homeafter discharge are important activities to reduce complications common complications after abdominal surgery are postoperative pulmonary complications andvenous thrombosis [ ] which generally are thought tobe partially avoidable with early mobilisationafter radical cystectomy for urinary bladder cancerthere is a high risk for postoperative complications thecomplications could be directly related to the patientshigh age to a high degree of comorbidity or both the major risk factor for developing urinary bladdercancer is smoking most of the patients are men andthe median age of undergoing a radical cystectomy is years [ ] as much as of patients are at severe nutritional risk before a radical cystectomy afterroboticassisted radical cystectomy rarc for urinarybladder cancer ofthe patients need to bereadmitted to hospital after discharge because of complications [ ]there is strong evidence that aerobic physical activityhas a positive impact on health survival and quality oflife qol patients diagnosed with cancer shouldfollow the general recommendations on physical activityand exercise for health [ ] yet most patients areinsufficiently active consequently with an increasing number of cancer survivors the importance of supporting high physical function and qol increases research has shown that exercise has a positive effect onhealthrelated qol hrqol in patients who have completed active cancer treatment moreoverin patients living with or beyond a diagnosis of cancerbehaviouraleg goalsetting andgraded tasks are important components in the exerciseinterventions with high adherence and positive physicaloutcomes support methodsin a recent study we evaluated the activity board®phystec sweden as a method to enhance mobilisationand recovery after abdominal surgery for cancer theactivity board is a tool based on techniques to supportbehaviour change [ ] the evaluation showed thatthe activity board resulted in a higher level of mobilisation in the group with the activity board compared withthe group who received standard treatment although evidence for exercise after abdominal surgery isscarce a few studies have evaluated exercise programmes for patients postoperatively atthe hospitalward with promising results [ ]despite the lack of exercise interventions after surgeryit has been shown that functional performance after aradical cystectomy for urinary bladder cancer correlatesto overall survival a large proportion of patientswith urinary bladder cancer do not achieve the recommendations on physical activity and exercise it isalso common that patients who undergo radical cystectomy have not performed physical exercise for a longtime before surgery finally after surgery patientsreport a low level of physical exercise recently tworeviews have been published on physical and psychological interventions to improve healthrelated outcomesin this patient group [ ] both reviews include thesame two postoperative exercise studies [ ] onelarger rct showed that early physical exercise and enhanced mobilisation after radical cystectomy positivelyaffected some domains of hrqol in addition in apilot study we tested a model for physical rehabilitationafter radical cystectomy the model consisted of weeks of individually tailored exercise after dischargefrom the hospital the exercise programme conductedat the hospital showed both short and longterm effectson physical function and hrqolconsequently the few studies within the field raiseseveral research questions for future exercise interventions in patients with urinary bladder cancer undergoingradical cystectomy current recommendations proposethe following areas supervised exercise after dischargethe optimal type of exercise fidelity and adherence ofthe intervention if shortterm outcomes are sustainedclinical relevance longterm outcomes and readmissionsto hospital [ ] we also need to understand thekinds of support that are optimal use behaviour change 0cporserud bmc cancer page of screened for eligibility in medical records by the responsible researcher rr and given written information by a registered nurse at a preoperative meetingafter days the rr will phone the patient provideoral information and then ask about participation informed consent will be signed before surgery the rrkeeps a protocol for enrolment the patient flowchartis depicted in fig eligibility criteriainclusion criteria will be patients who are planned for ararc for urinary bladder cancer the patients shouldbe able to talk and understand swedish without an interpreter be mobile with or without a walking aid and livein the stockholm regionstrategies and implement the intervention as a part ofthe patients clinical pathway through the healthcare system [ ]in summary patients who have been treated forurinary bladder cancer are not sufficiently physicallyactive and suffer from readmissions to hospital due tocomplications therefore there is a need for developing a physical rehabilitation programme to supportpatients who have a radical cystectomy in the earlypostoperative period in this paper we present a studyprotocol for the canmore trial a physical rehabilitation programme after rarc for urinary bladdercancerimpact ofamethodsdesignmain objectiveis to evaluatethe main aim of the canmore trialthestandardised and individuallyadapted exercise intervention in primary health carephcfunction primary outcome and habitual physical activity hrqol fatiguepsychological wellbeing and readmissions due tocomplications in patients undergoing rarc for urinary bladder cancerto improve physicalhypothesiswe hypothesise that the canmore programme is morebeneficial than homebased exercises in increasing physical function primary outcometrial designthe canmore trialis a randomised controlled trialrct with a singleblinded design the interventiongroup will receive a 12week h twice a week standardised and individually adapted exercise intervention inphc and behavioural support for daily physical activitythe control group will receive a homebased exerciseprogramme as well as recommendations on daily physical activity based on general guidelines we will followthe spirit standard protocol items recommendationsfor interventional trials statement and guidelinesfor reporting the study protocol the clinical trial registration number for this trial is nct03998579study settingthe study will be conducted in two settings a universityhospital and a phc context in region stockholmrecruitment and screeningparticipants will be recruited through theme canceratthe karolinska university hospital solna andscreened for eligibility recruitment will be performedconsecutively based on power analysis patientswill be included potential participants will initially befig patient flowchart 0cporserud bmc cancer page of exclusion criteriapatients with planned palliative surgery or cognitive impairment identified by screening of medical records willnot be includedrandomisation assignment of interventionpatients who fulfil the criteria for inclusion will afterhaving given their oral and written consent be randomised in the alea system operated by the clinical trials office cto atthe centre for clinical cancerstudies theme cancer karolinska university hospitalsolna swedenrandomisation will be conducted in blocks of patients stratified by sex and age ¥ years a confirmation email will be sent to the entering investigatorand an enrolment log will be filed at the centre the patient will receive the next consecutive code number inthe trial and treatment arm according to the randomisation schemeislogic model for the canmore programmeit is recommended that programme design should bebased on a theory to improve evidence synthesis the theoretical framework underpinning the canmoreprogrammethe movement continuum theorymct and the evidencebased calore taxonomy forbehavioural change techniques the mct posits that anindividual has three stages of movement capability amaximum a current and a preferred the canmoreprogramme identifies the patients current physical function capability and intervenes regarding the patientsneed for function several models and theories are supporting a behaviour change which results in increasedphysical activity however research has shown thatit is most often not necesssary for a complete theorybut instead the different components in the theory thatsupport the behaviour to consider the patients need forsupport the calore taxonomy for behavioural changetechniques has been added the taxonomy is recommended to be used to improve the specification of interventions behavioural techniques proven effective tosupport behaviour change are goalsetting graded tasksselfmonitoring feedback and reward all of these areused in the canmore programmea conceptual framework visualising the inputs theoryintervention components and its intermediate and possible longterm outcomes is depicted in a logic modelfig interventionall patients receive preoperative information on the importance of early mobilisation and postoperative individual physiotherapy during the hospital stay the activityboard is used for enhanced mobilisation before discharge from the hospital the patients receive standardised information about avoiding the lifting of heavyobjects and the importance of physical activity the patients are then randomised to either the intervention orthe control groupintervention grouppatients in the intervention group get a referral to aphysiotherapist in phc close to where they live the patients can choose from phc settings spread throughout the stockholm region within the third week afterdischarge the patients begin weeks of biweekly exercise the patients pay for their primary care visits physiotherapists in the targeted primary care units receive afig logic model of the intervention 0cporserud bmc cancer page of leaflet and a short education before starting comprisinginformation about rarc restrictions potential adverseevents the trial process and the exercise programmethe physical exercise is individually targeted but basedon international recommendations for persons with cancer disease the exercise programme includes aerobicexercise aiming at moderate intensity minsessionand strengthening exercises comprising endurance training with à repetitions see additional file theprogramme is gradually increased based on the patientscapability the programme also includes exercises forabdominal muscles including pelvic floor exercises tominimise the risk of developing stoma hernia however to avoid heavy strain on the surgery wounds restrictions regarding abdominal muscles are followed weeks postoperatively the exercise programme hasbeen approved by the responsible medical surgeons inaddition to the structured exercise sessions the patientsare advised to take daily walks in their neighborhoodthe number of recommended steps per day is set together with the physiotherapist based on the patientscapability to support the patient individual goalsettingfeedback and selfmonitoring of daily steps are usedsimilarly to those of the activity board at the end ofthe exercise periodthe physiotherapist recommendscontinued physical activity according to their clinicalroutinesanasapplicationactivitydiarypedometeroraphoneoutcomesthe measurements will be conducted using validated instruments at discharge ie baseline postintervention months and year after surgery table with thepurpose to receive information on the patients healthand physical function before surgery eg to adjust forin the analysis measurements will also be conductedbefore surgery all measurements will be conducted byexperienced physiotherapist blinded to the interventiona protocol for the measurements has been developedand the physiotherapist will receive specialised trainingby the research staffprimary outcomephysical function the primary outcome will be measured using the validated sixminute walk test 6mwt[ ] the test reproduces activity of daily living at asubmaximal level which is particularly applicable to elderly patients the patients are asked to walk as faras possible for min the number of meters m oxygensaturation and heart rate measured with a pulse oximeter will be recorded at the end of the test according tostandard procedures the primary outcome variablewill be walking distance in meterscontrol groupthe control group will receive oral and written information of a gradually increased homebased exerciseprogramme that includes daily walks and sittostandexercises they will also receive information on supportive techniques to improve physical activity suchsecondary outcomesgait speed will be assessed with the 10m walk test the test is used to determine walking speed in metersper second ms over a short distance the test is performed as three 10m walks without assistance one testwalk one in preferred walking speed and one in thepreop testingxbaselinexposttestingx1year followupxxxxxxxxxxxxxxxxxxxxxxxxxxxxxtable outcome measures and test occasionsvariablephysical functionmeasure6min walk testgait speedleg strength10m walk testchair stand testhandgrip strengthjamar hand dynamometerhabitual physical activityprevious physical activity levelahealthrelated quality of lifefatiguepsychological wellbeingpainactivpal3 microstanford brief activity surveyeortc qlqc30eortc qlqblm30piper fatigue scalehadsnrslength of hospital stay dayscomplicationsmedical recordsmedical recordsxxxxxxxxreadmissionsaprevious physical activity level is only used for adjustment purposes in the analysismedical records 0cporserud bmc cancer page of fastest speed possible time is measured for the intermediate m to allow for acceleration and decelerationthe outcome variable is msgrip strength will be assessed with the validated jamarhydraulic hand dynamometer the patients will sitin a chair and hold the dynamometer the test is performed three times for each hand and a mean value foreach hand is calculated the outcome variable is the gripstrength reading in kgleg strength will be assessed with the 30s sec chairstand test the patient is asked to rise from a chairas many times as possible for s the outcome variableis the number of sittostand transitionshabitual physical activity will be measured usingthe activpal3 micro activity monitor pal technologies ltd glasgow uk [ ] the activpal is asmall device which when attached to the thigh provides information based on position and accelerationof the body the information is then transferred tobody posture the transition between postures stepping and stepping speed the activity monitor is attached to the anterior midline ofthe thigh withdressing and does not provide feedback to the patientthe monitor will be worn for seven consecutive daysafter discharge from hospital and after the intervention period outcome variables will be time spentsittinglying standing stepping numbers of stepcounts and sittostand transitionsselfreported previous physical activity level will bemeasured using the 2item stanford brief activitysurvey sbas the sbas assesses the usualamount and intensity of physical activity during thepast year that a person performs the first item describes five patterns of work activity ranging frommostly sedentary to hard physical labour the seconditem describes five patterns ofleisuretime physicalactivity ranging from sedentary to regular vigorousintensity aerobic activities for respondents who areretired and have no job or regular work they wouldselect the response not applicable for both itemsthe outcome variable was categorical and rated on a5point scalehrqol will be assessed using the eortc qlqc30 with addition of the eortc qlqblm30 questionnaire the eortc qlqblm30 is explicitlydeveloped for patients with muscleinvasive urinarybladder cancerfrom theworst to the best for functional health statusand from the best to the worst for symptoms outcome variables will range from to fordifferent domains scoring rangesfatigue will be assessed using the piper fatigue scale the questionnaire consists of items and scoringranges from noneto severe the score ispresented in four domains plus a total fatigue scoreoutcome variables will range from to psychological wellbeing will be assessed using the hospital anxiety and depression scale hads thescale consists of questions with each scored on ascale from to where represents more symptomsthe questions are equally divided into the domains anxiety or depression each domain can result in a maximum score of outcome variables will range from to pain will be assessed using the numeric rating scalenrs the nrs is an eleven point scale and scoring ranges from no pain to worst pain the nrsis verbally delivereddata on length of stay at the hospital and frequency ofreadmission to hospital due to complications will be extracted from patient medical records readmissions willbe extracted as and days after surgery and complications will be registered using the claviendindo classification [ ]ethicsthe project is approved by the regional board of ethicsin stockholm dnr and the swedishethical review authority dnr the new model for rehabilitation is compared withsimilar care the patients are given in todays care delivery yet the control group will receive less attentionthan the intervention group we find it unethical to askthe patients to come to phc pay their visit and only receive for example stretching exercises in addition itwill be challenging to motivate the physiotherapists inphc to offer such treatmentsample sizefunction evaluatedthe primary outcome is physicalwith the validated 6mwt based on data from our pilotstudy we calculate an increase in m in theintervention group m in the control group and astandard deviation of m to obtain a statistical powerof with a type error set at patients areneeded in each group however as the test is highlycorrelated with sex and age we will stratify the analysis and increase the sample sizefor the secondary outcome readmissions due to complications we will estimate readmissions in theintervention group compared with the proportion of patients being readmitted today which is in the control group to obtain a statistical power of with atype error of patients are needed in eachgroup taken all this into account and guard againstdropout patients in each group will be includedin the study 0cporserud bmc cancer page of data management and study databasedata will be entered using an electronic system pheedit which is based on the sas system provided and operated by the cto at the centre for clinical cancerstudies theme cancer karolinska university hospitalsolna sweden a data management plan is delivered bycto documenting the database and all procedures fordata management the investigator verifies that all dataentries in the case report forms crfs are accurate andcorrect if certain assessments according to the protocolare not performed for any reason or if certain information is not available not applicable or unknown this willbe indicated in the crf by the investigator the investigator is required to sign off all reported datasource datain this study physicaltests movement sensor datapatientreported outcome measures and medical recordswill be regarded as source datacodingin the study database patients will be identified onlythrough the unique randomisation number all data willbe stored with coded identification and no access to thepatients id patient identification will not be revealed intext files logbooks with identification numbers and therespective codes will be stored in a locked environmentat the local centre that is not accessible to personnel notinvolved in conducting the trialstatistical analysisdescriptive statistics will be performed to ensure comparability between data at baseline to evaluate the effectof the intervention mixed models or linear regressionmodels spss inc chicago il usa according to theintention to treat procedure and with an alpha level of will be used significance of main or interaction effects will be explored using the bonferroni posthoc multiple comparison test in the case of skewed distributionlogarithmic transformations or corresponding nonparametric statistics will be used to assess the effect of theinterventionimplementation processbecause the intervention design is intricate we will inaddition to testing effects of the canmore programmeon patientlevel outcome measures also observe andgather information on factors that might have influencedthe implementation of the programme the evaluation of the implementation process will be based on themedical research council guide for process evaluationof complex interventions the knowledge gainedcan also be used to offer recommendations on whichstrategies to use when implementing the canmoreprogramme in other clinical settings and on a largescalethe initial strategies for the process evaluation will include meetings with surgeons the head of the surgicalward and phc clinics discussions and involvementwith physiotherapists and nurses at the surgical wardand physiotherapists at phc clinics and education ofthe canmore programme and outcome measures tophysiotherapists who will be involved in the intervention a leaflet for the patients an extended educationalleaflet and a short education for the physiotherapistshave been developedto study what is delivered measures of fidelity doseadaptation and reach will be assessed fidelity relative tothe canmore programme will be evaluated as the extentto which the programme was delivered as expecteddose will be assessed as the quantity of the interventionthe canmore programme and the education of physiotherapists in phc implemented adaptation such aschanges done to fit different phc settings will be reported in a questionnaire reach will be assessed regarding how many eligible patients signed an informedconsent form and how many in the intervention groupfulfilled the canmore programme in addition adverseevents will be registerediethe extentcontext includes external factors that may act as abarrier or facilitator to both implementation itself andthe patient level effect assessing barriers and facilitators to programme implementation will also involveevaluating programme feasibilitytowhich patients and health care staff regard the canmore as satisfactory in terms of content and complexitydifficulty we plan to conduct an interviewstudy on patients experiences of the intervention inaddition we plan to collect information on possiblebarriers that might have influenced the implementation ofthe programme and facilitators that mighthave supported it at the various clinical sites bothqualitative structured observationfocus groups andindividual semistructured interviews and quantitativequestionnaires and enrolment files methods will beused to assess how the intervention was delivered aswell as experiences of the different stakeholders patients physiotherapists and other health care staff aswell as managersby using several sources for data collection triangulation can be achieved which supports the trustworthinessof the study the consolidated framework for implementation research cfir will be used in the currentstudy to guide the investigation of context ie potentialbarriers and facilitators of the implementation processconstructs that we believe specifically impact the implementation outcomes in the present study and guidelinesforand observation protocolsinterview questions 0cporserud bmc cancer page of published by cifr will be followed httpcfirguidetoolshtmldiscussionto our knowledge this is the first study to examine theeffects of individually targeted exercise in phc compared with traditional advice on home exercise trainingafter rarc moreover the study will include a processevaluation of factors thought to influence the implementation of the programmealthough there is evidence that exercise is beneficialto improve physical function physical activity hrqolreduce fatigue and perhaps reduce complications it isessential to design feasible and easy to implement interventions in a health care setting our intervention is individually targeted and designed based on currentglobal guidelines for physical activity and exercise strategies for behavioural support and adapted to fitwithin the phc structure the length of the interventionis based on exercise principles and what is feasibleto conduct within phc yet this study does not tell uswhether the length of the intervention is the optimallength for best health benefits nor if a booster sessionafter the intervention period is needed the dose of exercise has been previously tested in a pilot study andshowed a positive effect on physical function was safeand had no adverse events after the pilot study werevised the exercise programme to be individually basedbut still include aerobic and musclestrengthening exerto ourcises the addition of behaviourprogrammeselfmonitoring and feedback has been shown to be associated with increased physical activity behaviour setting graded taskseg goalsupportat discharge from the hospital the standard care forpatients includes information on the importance ofphysical activity in this study the control group receivea light intervention ie recommended daily walks andlegstrengthening exercises instead of the standard carethe exercise recommendation is low dose and not specific but can still affectthe results by producing asmaller difference between the groups because there isstrong evidence for the effect of physical activity we findit unethical not to give the control group any advice onphysical activity at discharge many patients are feeblebecause of surgery and the postoperative period at hospital which can also result in fear of movement thesepatients require supervised physical exercise as in theintervention group in this studythe process evaluation using measures offidelitydose adaption adherence and reach as well as patientperspectives and experiences of the programme can helpexplain the results in additionit can speed up theprocess of translating findings from research settings toclinically representative settings the programme fitswell within the healthcare system and the exercises aregeneric to those recommended for cancer survivors andthe tools for motivational support are generic for thewhole population if the programme is proven effectiveit should be generalisable to other patient groupsthatis notthere are some limitations first due to the difficultiesof doubleblinding we have a singleblind design inwhich a physiotherapistinvolved in theprogramme is conducting the measurements secondwe foresee a long recruitment period that can lead to achange in health care staffphysiotherapists in phc toensure quality we plan to have continued contact withthe clinics and a new educational structure for trainingif neededcystectomyin summary this proposed rct has the potential toprovide new knowledge within rehabilitation after radicalcancer theprogramme should be readily applied to other patientgroups undergoing abdominal surgery for cancer andhas the potential to change the health care chain forthese patientsfor urinary bladdersupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071405additional file exercise programmeabbreviationscanmore cancer mobilisation rehabilitation cfir consolidated frameworkfor implementation research crf case report form cto clinical trials officehads hospital anxiety depression scale hrqol healthrelated quality of lifemct movement continuum theory ms meters per second phc primaryhealth care qol quality of life rarc roboticassisted radical cystectomyrct randomised controlled trial rr responsible researcher sbas stanfordbrief activity survey sec seconds spirit standard protocol itemsrecommendations for interventional trials 6mwt sixminute walk testacknowledgementsnot applicableauthors contributionsmh and ap conceived the idea for this study and designed it along with theother authors all authors ap pk er ma lh mnb and mh were involvedin drafting and revising the manuscript all authors will be involved in datacollection analysis or manuscript preparation as the study proceeds allauthors read and approved the final manuscri | 0 |
"Previous evidence has suggested that lower gestational vitamin D levels might increase the risks ofadverse pregnancy and birth outcomes The results remain inconsistent and require further explorationMethods A total of Chinese motherinfant pairs were included in this retrospective cohort study Serumconcentrations of 25OHD were reviewed in early pregnancy ± weeks Outcomes of maternal gestationaldiabetes mellitus GDM cesarean section fetal distress preterm birth low birth weight LBW and macrosomiawere extracted from the medical records Cox regression analysis was used to explore these associationsResults In total of mothers were pregnant at an advanced age ¥ years and of pregnant womenhad vitamin D deficiency nmolL After adjusting for potential covariates the hazard ratio HR CI perstandard deviation SD increase of serum 25OHD concentrations was for GDM for preterm birth and for LBW Similar protective associations were found for GDMcesarean section and preterm birth for a better vitamin D status when compared with vitamin D deficiencyConclusion Higher early pregnancy vitamin D was associated with a lower risk of GDM cesarean section pretermbirth and LBWKeywords 25ohd Vitamin D Pregnancy Maternal outcome Infant outcomeBackgroundVitamin D is a secosteroid hormone that is well knownfor its physiological function in maintaining bone metabolism and health A high prevalence of vitamin D deficiencyusually defined as serum 25OHD levels nmolL hasbeen found in pregnant women globally especially in developing countries [] including China [ ] Increasing evidence has suggested that vitamin D sufficiency is important Correspondence liuzphlk81outlookcom Gengdong Chen and Tingting Pang contributed equally to this work1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong ChinaFull list of author information is available at the end of the for the prevention of pregnancy complications in mothersand adverse fetal birth outcomes [ ] although divergentresults have been reported in other studies [ ] Severalsystematic reviews based on randomized clinical trials orobservational studies have suggested that lower vitamin Dstatus contribute to adverse outcomes such as preeclampsia[] gestational diabetes mellitus GDM [ ] low birthweight LBW [] and preterm birth [] However increasing evidence shows different associations [ ]and no definitive has yet been made [] Severalproblems remain to be solved by further studies the heterogeneity of associations from diverse areas and populations with different vitamin D status serve as evidencethewhen makingguidelinesregionsforspecific The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Pregnancy and Childbirth Page of observation or supplementation of vitamin D in the thirdtrimester in many studies might present the problem ofcausal inference or miss the practical period for the intervention In addition the results from early trimesters mightbe helpful and more important for the early prevention ofadverse outcomesIn additionit has been suggested that the risk ofadverse complications or birth outcomes increases asmaternal age increases [] With the change ofpopulation policy in China the percentage of womenpregnant at an advanced age years has increasedand proper strategies are urgently needed for the prevention of adverse complications [] However whetherthe influence of vitamin D on maternal and infant outcomes remained the same for women pregnant at young years and advanced ages remains unclear andmore studies are needed to better illustrate the problemWe investigated the relationship between early gestational serum 25OHD concentrations and several adverse maternal and infant outcomes in a retrospectivecohort study including Chinese motherinfantpairs Our results provide further evidence for clinicalrecommendations on the early prevention of related adverse outcomes in this fieldMethodsThe study used data that were gathered from a largecenter Affiliated Foshan Maternity Child HealthcareHospital Southern Medical University Foshan CityGuangdong Province China from September toJuly The hospital is the largest gynecology andobstetrics center in Foshan City and covers a large population of million people The included subjects werewomen who underwent early pregnancy serum vitaminD measurement gestational weeks and deliveredtheir infants at the hospital The exclusion criteria included twin or higherorder multiple pregnancies seriousdiseases such as type diabetes mellitus cardiovasculardiseases thyroid disorder and cancer that occurred before pregnancy Ultimately a total motherinfantpairs were included in this study The study was approvedby the ethics committee of Affiliated Foshan Maternity Child Healthcare Hospital Southern Medical UniversityData collectionVitamin D data from the clinicallaboratory werereviewed Blood samples were collected during the regular obstetric checkups and immediately measured by aclinical laboratory without being frozen Serum concentrations of 25OHD 25OHD2 and 25OHD3 weredetected using colloidal gold immunochromatographyCommercial kits were obtained from Mei Ning KangCheng Bio Tec Inc The intraassay and interassay coefficients of variation were less than Outcomes including GDM cesarean section fetal distress preterm birth low birth weight and macrosomiawere extracted from medical records and reexamined bytwo independent staff members The disease diagnoseswere made by professional doctors with the samestandardization criteria and were extracted from themedical records Gestational hypertension preeclampsiaor eclampsia was not included because of the lack ofavailable cases An oral glucose tolerance test was performed from to gestational weeks and GDM wasdiagnosed if the subjects met any of the following criteria fasting blood glucose ¥ mmolL onehour bloodglucose postoral sugar ¥ mmolL or twohour bloodglucose postoral sugar ¥ mmolL Preterm birth wasdefined as delivery at ¥ but gestational weeksLBW was diagnosed if the neonatal birth weight g while a neonatal birth weight ¥ g was defined asmacrosomia Other variablesincluding the maternalage body mass index BMI gestational age parity season of blood collection and time of delivery were alsoextracted from the medical recordsStatistical analysesContinuous variables were represented by the mean ±standard deviation SD or median interquartile rangeand tested by Students ttest Categorical variables wererepresented by frequencies percentage and tested bythe chisquare test Cox regression analysis was performed to explore the associations between vitamin Dand maternal or infant outcomes Serum concentrationsof 25OHD 25OHD2 and 25OHD3 were first Zstandardized before being included in the regressionVitamin D deficiency was defined as serum 25OHDlevels of nmolL Two different models were testedwith Model as a univariate model without adjustmentand Model adjusted for maternal age BMI parity andseason the blood was collected and measured Stratifiedanalyses were performed according to the gestationalage young years advanced ¥ years All the analyses were performed using SPSS software version SPSS Inc Chicago IL USA A twosided P value ofless than was considered statistically significantResultsA total motherinfant pairs were included in thisstudy A high prevalence of gestational vitamin D deficiency was discovered in the mothers Even insubjects without vitamin D deficiency the highest serum25OHD concentration was only nmolL Compared with women pregnant at a younger age the subjects with advanced age of pregnancy ¥ years tendedto have a higher BMI parity higher percentage of vitamin D deficiency higher incidence of GDM cesareansection preterm birth and LBW but a lower gestational 0cChen BMC Pregnancy and Childbirth Page of age lower serum 25OHD and OHD3 concentrations lower incidence of fetal distress and lower incidence of macrosomia Table As shown in Table although the 25OHD concentrations were statistically higher in spring and summerthan those in autumn and winter only small differenceof 25OHD values to nmolL was observedbetween different seasons especially for 25OHD2 Significant protective associations were found between thevitamin D levels and GDM and preterm birth Afteradjusting for potential covariatesTable higher25OHD concentrations per one SD increase were associated with a HR CI decrease in the GDM risk a HR CI decrease in the preterm birth risk and a HR CI decrease in theLBW risk Similar protective results were also found for25OHD2 and 25OHD3 but the associations tended tobe more pronounced for 25OHD2 Null associationsbetween vitamin D and cesarean section fetal distressand macrosomia were observed in all the subjects Maternal serum 25OHD levels ¥ nmolL were associated with a decrease in the GDM risk a decrease in the cesarean section risk and a decrease in the preterm birth risk but the trend did nothold with the other outcomes compared with those withvitamin D deficiency Table After stratification by gestational age Table higherlevels of vitamin D were associated with a lowerrisk of GDM in those years In contrast a protectiveassociation of vitamin D with low birth weight wasfound for women pregnant at ¥ years but not years However no significant interactions were discovered Pinteraction Table Characteristic of subjectsAge yearsBMI kgcm2Gestational age weeksParity timesNeonatal birth weight kg25OHD nmolL25OHD2 nmolL25OHD3 nmolLVitamin D deficiency N YesNoGestational diabetes mellitus N YesNoCaesarean section N YesNoFetal distress in uterus N YesNoPreterm birth N YesNoLow birth weight N YesNoMacrosomia N YesNoTotal N ± years N ± ¥ years N ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P 0cChen BMC Pregnancy and Childbirth Page of Table Seasonal difference between serum vitamin D indicatorsDetected Seasonspring summer ± ± ± autumn winter ± ± ± P 25OHD nmolL25OHD2 nmolL25OHD3 nmolLDiscussionIn this retrospective cohort study including Chinese motherinfant pairs protective associations werefound for higher serum 25OHD concentrations withGDM cesarean section postpartum hemorrhage preterm birth and low birth weight but not for the otheroutcomes The results for 25OHD2 tended to be morepronounced than those for 25OHD3Interestinglyhigher serum vitamin D contributes to a higher risk ofpostpartum anemia in women pregnant at a young agebut not in advanced yearsIn this population with a high prevalence of vitamin Ddeficiency better serum vitamin D status contributed toa lower risk of GDM This result was consistent withseveral other studies A to higher risk of GDMwas observed for pregnant women with insufficient ordeficient vitamin D status in three systematic reviewsand metaanalyses based on observational studies orclinical trials [ ] In a large randomized controltrial RCT based on Iranian women the intervention of 25OHD3 was associated with lower risk ofGDM [] The results were further supported by severalother prospective cohort studies [ ] However nullassociations were found between the vitamin D status orsupplements and GDM in a systematic review based onfive randomized trials including subjects [] anested casecontrol study of women [] and alarge prospective study including motherchildpairs [] Additionally two studies found detrimentalassociations of higher vitamin D levels with GDM butthe effects were tiny [] or restricted to specificethnicities Hispanic []Consistent with our results the protective associationof maternal vitamin D status with a lower risk of preterm or low birth weight has also been reported in severalstudies An inverse doseresponse relation ofvitamin D status was found for preterm birth in a systematic review and metaanalysis based on longitudinal studies[] Rostami reported that a 25OHD3 interventionof monthly IU could be beneficial for the preventionof preterm delivery in an RCT of Iranian women []In a large prospective cohort including motheroffspring pairs the risk of low birth weight was CI and CI among subjects with vitamin D deficiency and insufficiency respectively [] Higher maternal serum 25OHD was positivelyTable Associations between early pregnant serum vitamin D concentrations and maternal infant outcomes25OHD aHR95CIPa25OHD2HR95CIPa25OHD3HR95CIPGestational diabetes mellitusModel Model Caesarean sectionModel Model Fetal distressModel Model Preterm birthModel Model Low birth weightModel Model MacrosomiaModel Model Cox regression analysis were operated for exploration of associations Model without adjustment Model adjusted for age BMI parity season of bloodcollected a Per one SD increase 0cChen BMC Pregnancy and Childbirth Page of Table Associations between vitamin D status and maternal infant outcomesGestational diabetes mellitusCaesarean sectionFetal distressPreterm birthLow birth weightMacrosomia25OHD nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolLHR95CI PCox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collectedassociated with higher birth weight in a study of UnitedArab Emirates [] and supplementation of vitamin D at adose of IUd was optimal and safe for mothers andtheir infants in United Arab Emirates [] However nullor detrimental associations were also reported in otherstudies Although vitamin D increased the mean birthweight it did not significantly reduce the risk of low birthweight or preterm birth in a metaanalysis based on randomized trials [] these results were further supported byanother update study [] In addition higher 25OHDconcentrations were found to increase the riskof preterm delivery in a prospective study of pregnant Chinese women []Gestational vitamin D deficiency was found to be associated with a 2fold increased risk of cesarean section ina cohort of lowincome pregnant women []Within a cohort of women from the United Statesfor women with 25OHD concentrations lower than nmolL the risk of a primary cesarean section wasalmost times higher [] These results were consistent with ours However a metaanalysis of trials subjects found null associations of vitamin D supplements with cesarean section []Much heterogeneity exists in the previous evidence inthis field and there may be several factors that partly explain these differences First most of the randomized trials included had a small sample size and were of lowquality Most subjects included in the trials had sufficient vitamin D status ¥ nmolL and a further doseof a or IUd supplement might have attenuatedTable Subclass analysis of relationship between serum vitamin D concentrations and related outcomes stratified by gestational ageP bP bP baa25OHD aHR95CIP25OHD2HR95CIP25OHD3HR95CIPGestational diabetesmellitusCaesarean sectionFetal distressPreterm birth y ¥ y y ¥ y y ¥ y y ¥ y Low birth weight y ¥ y Macrosomia y ¥ y Cox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collecteda Per one SD increase b P for interaction 0cChen BMC Pregnancy and Childbirth Page of the detrimental influence of vitamin D deficiency or insufficiency in the reference group [] Increasing benefitsmight not exist for higher doses Second the vitamin Dreceptor is important for the utilization of vitamin D[ ] The difference in vitamin D receptor gene polymorphism might help explain the ethnic heterogeneity[] This requires further confirmation in the futureThird the studies were conducted during different trimesters of gestation which might increase the difficultyof making comparisons those studies conducted duringan early gestational period might be advantageous forcausal inference and early preventionBecause 25OHD3 contributes most to the 25OHDconcentrations it has been used to represent 25OHDin many studies and only a few studies have focused on25OHD2 In our study the results were generally consistent between these two subcomponents The associations tend to be more pronounced in 25OHD2 than in25OHD3 and mightindicate the prominence of25OHD2 over 25OHD3 for the prevention of relateddiseases This theory merits further confirmation Nevertheless our results and others provide further evidencefor the importance of a better early vitamin D status forthe prevention of GDM cesarean section preterm birthand low birth weight in a population with a high prevalence of vitamin D deficiency Our results indicated thatmore consideration should be given to distinguishing between the vitamin D subcomponents women pregnantat different ages and women with more pregnancy complications in this field Considering the high prevalenceof vitamin D deficiency during pregnancy observed inChina [ ] it is a matter of urgency to call for the supplementation of vitamin D and more efforts should bemade to improve the gestational vitamin D status ofChinese populationVitamin D deficiency was found to decrease vasculardiameter within the labyrinth region and dysregulateplacental development during early pregnancy in an animal experiment [] Vitamin D supplementation duringearly pregnancy might rescue this situation while furthersupplementation might be futile after missing this timepoint [] Moreover vitamin D supplementation duringearly pregnancy is likely to affect genetic information ofsystemic inflammation and immune responses involvedin the development of gestational comorbidities egGDM preeclampsia and infection as reviewed by Hollis [] These mechanisms help to explain the beneficialinfluences of vitamin D for maternal and infanthealth and emphasize the importance of improving vitamin D status during early pregnancyOurstudy had several advantages Firstserum25OHD concentrations were measured during an earlygestational period and with the retrospective cohort design we assured the temporal sequence and avoided thepossibility of causal inversion Second we studied multiple outcomes and conducted further analysis of thesubcomponent of vitamin D and a stratified analysis ofgestational age which provided a more comprehensiveunderstanding of this field There are also several limitations of our study First our study was limited by thelack of information on dietary vitamin D intake including by supplement and sunlight exposure during thepregnancy period The obtained data were difficult touse for a retrospective study while the use of blood indicators might be more precise than a dietary survey sowe adjusted the association for different seasons to attenuate their influence Second the 25OHD concentration was measured only once we could not monitor thedynamic changes afterward or determine whether theywere normalized after receiving vitamin D supplementation However the associations tended to be underestimated instead of overestimated Third Wagner et al[] and Mirzakhani [] indicated a 25OHDconcentration ¥ nmolL in early pregnancy was optimal for the prevention of preterm birth and preeclampsia however the highest 25OHD concentration in ourstudy is only nmolL Therefore we were unable toperform the analyses using a threshold of nmolLand assess the influences of higher 25OHD concentrations Finally residual confounding might still existthough we tried to control several potential covariatesConclusionsThis retrospective cohort study showed the beneficial associations of early gestational vitamin D with outcomesof GDM cesarean section preterm birth and low birthweight More welldesigned randomized clinical trialsare needed for further exploration and confirmation ofthese resultsAbbreviationsGDM Gestational diabetes mellitus LBW Low birth weight BMI Body massindex SD Standard deviationAcknowledgementsWe would like to thank Ye Shaoxin Yang Xiaoming Liu Haojing Wangdong and Huang Shaobing for their generous help in this studyAuthors contributionsGDC and ZPL devised the idea and designed the study GDC TTP PSLZXZ DXL DZF contributed to the primary data collection GDC and TTP reexamined the data GDC TT P PS L ZX Z DXL DZF contributedto the analysis of the data GDC and TTP wrote the original draft whichwas revised by XLG and ZPL XLG and ZPL supervised the study andZPL administered the project All authors have read and approved themanuscriptFundingThis work was supported by the Basic and Applied Basic ResearchFoundation of Guangdong Province No 2019A1515110163 GDC and theFoundation of Bureau of Science and Technology of Foshan City No GDC The funding sponsors had no role in the design ofthe study in the collection analyses or interpretation of data in the writingof the manuscript and in the decision to publish the results 0cChen BMC Pregnancy and Childbirth Page of Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author upon reasonable requestEthics approval and consent to participateThe study was approved by the ethics committee of Affiliated FoshanMaternity Child Healthcare Hospital Southern Medical University TheAffiliated Foshan Maternity Child Healthcare Hospital providedadministrative permissions for the research team to access and use the dataincluded in this research Data were extracted from medical records and theconsent to participate was unavailable due to the retrospective design of thestudy and difficulty in reconnection however the private information waswell protectedConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong China 2Department of Medical RecordsAffiliated Foshan Maternity Child Healthcare Hospital Southern MedicalUniversity Foshan Guangdong ChinaReceived February Accepted August ReferencesSaraf R Morton SM Camargo CA Jr Grant CC Global summary of maternaland newborn vitamin D status a systematic review Matern Child NutrYun C Chen J He Y Mao D Wang R Zhang Y Yang C Piao J Yang XVitamin D deficiency prevalence and risk factors among pregnant Chinesewomen Public Health Nutr Zhou J Su L Liu M Liu Y Cao X Wang Z Xiao H Associations between hydroxyvitamin D levels and pregnancy outcomes a prospectiveobservational study in southern China Eur J Clin Nutr Agarwal S Kovilam O Agrawal DK Vitamin D and its impact on maternalfetal outcomes in pregnancy a critical review Crit Rev Food Sci Nutr von Websky K Hasan AA Reichetzeder C Tsuprykov O Hocher B Impact ofvitamin D on pregnancyrelated disorders and on offspring outcome JSteroid Biochem Mol Biol Nobles CJ Markenson G ChasanTaber L Early pregnancy vitamin D statusand risk for adverse maternal and infant outcomes in a biethnic cohort thebehaviors affecting baby and you BaBY study Br J Nutr Roth DE Leung M Mesfin E Qamar H Watterworth J Papp E Vitamin Dsupplementation during pregnancy state of the evidence from a systematicreview of randomised trials BMJ 2017359j5237HautaAlus HH Viljakainen HT HolmlundSuila EM EnlundCerullo MRosendahl J Valkama SM Helve OM Hytinantti TK Makitie OM AnderssonS Maternal vitamin D status gestational diabetes and infant birth size BMCPregnancy Childbirth Khaing W Vallibhakara SA Tantrakul V Vallibhakara O Rattanasiri S McEvoyM Attia J Thakkinstian A Calcium and vitamin D supplementation forprevention of preeclampsia a systematic review and network metaanalysisNutrients 2017910E1141 Zhang Y Gong Y Xue H Xiong J Cheng G Vitamin D and gestationaldiabetes mellitus a systematic review based on data free of Hawthorneeffect BJOG Zhang MX Pan GT Guo JF Li BY Qin LQ Zhang ZL Vitamin D deficiencyincreases the risk of gestational diabetes mellitus a metaanalysis ofobservational studies Nutrients Aghajafari F Nagulesapillai T Ronksley PE Tough SC O'Beirne M Rabi DMAssociation between maternal serum 25hydroxyvitamin D level andpregnancy and neonatal outcomes systematic review and metaanalysis ofobservational studies BMJ 2013346f1169 Qin LL Lu FG Yang SH Xu HL Luo BA Does Maternal Vitamin D DeficiencyIncrease the Risk of Preterm Birth A MetaAnalysis of Observational StudiesNutrients 201685E301 Rodriguez A GarciaEsteban R Basterretxea M Lertxundi A RodriguezBernalC Iniguez C RodriguezDehli C Tardon A Espada M Sunyer J et alAssociations of maternal circulating 25hydroxyvitamin D3 concentrationwith pregnancy and birth outcomes BJOG Ogawa K Urayama KY Tanigaki S Sago H Sato S Saito S Morisaki NAssociation between very advanced maternal age and adverse pregnancyoutcomes a cross sectional Japanese study BMC Pregnancy ChildbirthKhalil A Syngelaki A Maiz N Zinevich Y Nicolaides KH Maternal age andadverse pregnancy outcome a cohort study Ultrasound Obstet GynecolJolly M Sebire N Harris J Robinson S Regan L The risks associated withpregnancy in women aged years or older Hum Reprod Li Q Deng D New medical risks affecting obstetrics after implementation ofthe twochild policy in China Front Med Poel YH Hummel P Lips P Stam F van der Ploeg T Simsek S Vitamin Dand gestational diabetes a systematic review and metaanalysis Eur J InternMed Rostami M Tehrani FR Simbar M Bidhendi Yarandi R Minooee S Hollis BWHosseinpanah F Effectiveness of prenatal vitamin D deficiency screeningand treatment program a stratified randomized field trial J Clin EndocrinolMetab Xu C Ma HH Wang Y Maternal early pregnancy plasma concentration of25Hydroxyvitamin D and risk of gestational diabetes mellitus Calcif TissueInt Boyle VT Thorstensen EB Mourath D Jones MB McCowan LM Kenny LCBaker PN The relationship between 25hydroxyvitamin D concentration inearly pregnancy and pregnancy outcomes in a large prospective cohort BrJ Nutr Schneuer FJ Roberts CL Guilbert C Simpson JM Algert CS Khambalia AZTasevski V Ashton AW Morris JM Nassar N Effects of maternal serum hydroxyvitamin D concentrations in the first trimester on subsequentpregnancy outcomes in an Australian population Am J Clin Nutr Amegah AK Klevor MK Wagner CL Maternal vitamin D insufficiency andrisk of adverse pregnancy and birth outcomes a systematic review andmetaanalysis of longitudinal studies PLoS One 2017123e0173605 Chen YH Fu L Hao JH Yu Z Zhu P Wang H Xu YY Zhang C Tao FB XuDX Maternal vitamin D deficiency during pregnancy elevates the risks ofsmall for gestational age and low birth weight infants in Chinesepopulation J Clin Endocrinol Metab Amirlak I Ezimokhai M Dawodu A Dawson KP Kochiyil J Thomas LAbdulle AM Current maternalinfant micronutrient status and the effects onbirth weight in the United Arab Emirates East Mediterr Health J Dawodu A Saadi HF Bekdache G Javed Y Altaye M Hollis BW Randomizedcontrolled trial RCT of vitamin D supplementation in pregnancy in apopulation with endemic vitamin D deficiency J Clin Endocrinol Metab PerezLopez FR Pasupuleti V MezonesHolguin E BenitesZapata VA Thota PDeshpande A Hernandez AV Effect of vitamin D supplementation duringpregnancy on maternal and neonatal outcomes a systematic review and metaanalysis of randomized controlled trials Fertil Steril 1288e1274Scholl TO Chen X Stein P Maternal vitamin D status and delivery bycesarean Nutrients Merewood A Mehta SD Chen TC Bauchner H Holick MF Associationbetween vitamin D deficiency and primary cesarean section J ClinEndocrinol Metab Chun RF Peercy BE Orwoll ES Nielson CM Adams JS Hewison M VitaminD and DBP the free hormone hypothesis revisited J Steroid Biochem MolBiol Pt A132 Bikle DD Gee E Halloran B Kowalski MA Ryzen E Haddad JG Assessmentof the free fraction of 25hydroxyvitamin D in serum and its regulation byalbumin and the vitamin Dbinding protein J Clin Endocrinol Metab Powe CE Evans MK Wenger J Zonderman AB Berg AH Nalls M Tamez HZhang D Bhan I Karumanchi SA Vitamin Dbinding protein and 0cChen BMC Pregnancy and Childbirth Page of vitamin D status of black Americans and white Americans N Engl J MedLiu NQ Ouyang Y Bulut Y Lagishetty V Chan SY Hollis BW Wagner CEquils O Hewison M Dietary vitamin D restriction in pregnant female miceis associated with maternal hypertension and altered placental and fetaldevelopment Endocrinology Mirzakhani H Litonjua AA McElrath TF O'Connor G LeeParritz A Iverson RMacones G Strunk RC Bacharier LB Zeiger R Early pregnancy vitaminD status and risk of preeclampsia J Clin Invest Hollis BW Wagner CL Vitamin D supplementation during pregnancyimprovements in birth outcomes and complications through directgenomic alteration Mol Cell Endocrinol Wagner CL Baggerly C McDonnell SL Baggerly L Hamilton SA Winkler JWarner G Rodriguez C Shary JR Smith PG Posthoc comparison ofvitamin D status at three timepoints during pregnancy demonstrates lowerrisk of preterm birth with higher vitamin D closer to delivery J SteroidBiochem Mol Biol Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 2 |
This study was performed assess the clinical outcomes of elderly patients withosteoporotic femoral neck fractures FNFs AOOTA 31BC treated by initial uncementedtotal hip arthroplasty UTA or cemented total hip arthroplasty CTAMethods This study involved consecutive elderly patients with osteoporotic FNFs AOOTA31BC treated by initial UTA or CTA in our medical centre from to The primaryoutcomes were the Harris hip score HHS and the rates of revision loosening periprostheticfracture and dislocationResults In total patients were included in the final analysis UTA n¼ CTA n¼ The mean followup duration was months range months The mean HHS was1Department of Microsurgery Trauma and Hand SurgeryThe First Affiliated Hospital Sun Yatsen UniversityGuangzhou China2Department of Pediatrics The First Affiliated HospitalSun Yatsen University Guangzhou China3Department of Orthopaedics The First AffiliatedHospital Sun Yatsen University Guangzhou China4Department of Orthopaedics The Third PeoplesHospital of Wuxi Jiangsu Province The Affiliated Hospitalof Jiangnan University Wuxi China5Department of Urology The First Affiliated Hospital SunYatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsJunxing Ye Department of Orthopeadics The ThirdPeoples Hospital of Wuxi Jiangsu Province The AffiliatedHospital of Jiangnan University No Xingyuan NorthRoad Liangxi District Wuxi Jiangsu ChinaEmail yejunxing0514163comJintao Zhuang Department of Urology The First AffiliatedHospital Sun Yatsen University No Zhongshan 2ndRoad Yuexiu District Guangzhou ChinaEmail brianzg86163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical Research 06 for UTA and 06 for CTA Significant dissimilarities were detected in therates of revision loosening and periprosthetic fracture between UTA and CTA vs vs and vs respectively A significant difference was also detected inthe probability of revision between the two groupsConclusion Elderly patients with osteoporotic FNFs AOOTA 31BC treated with CTA showgreater improvements in functional outcomes and key orthopaedic complications than thosetreated with UTAKeywordsFemoral neck fracture arthroplasty outcome complication retrospective osteoporosisDate received December accepted July Introduction31BCAOOTAfemoral neck fracturesManagement ofin elderlyFNFspatients is still undergoing considerableresearch12 Uncemented total hip arthroplasty UTA or cemented total hip arthroplasty CTA for displaced FNFs tendsto be a recognised surgical strategy3Comparisons between UTA and CTA forelderly individuals with an FNF generallyfavour CTA this is primarily attributed tothe exceptional clinical outcomes of CTA interms of relieving pain and improving dailyactivities as well as the higher rate of majororthopaedic complications ie revisionloosening periprosthetic fracture and dislocation associated with UTA4 Howeverrecent studies of UTA in elderly individualshave demonstrated encouraging shorttermclinical outcomes67 Moreover cementedprosthesis syndrome tends to occur morefrequently in CTA than UTA8 Cementedprosthesis syndrome theoretically poses asignificantlifealthough the speciï¬c probability of thisthreat has not been calculated89 Hencewhether to utilise CTA for elderly individuals may present the clinician with a dilemma9 The lack of consensus regarding whichtechnique UTA or CTA is preferable fortreating FNFs AOOTA 31BC in elderlyindividuals is related to the remarkableto the patientsthreatdistinction in clinical outcomes betweenthe two types of implants610Most previousconcentrated medicalstudies have involvedhighlycentresand several brands of prostheses356Additionally shortterm followup is commonplace in these studies To overcomethese drawbacks of previous studies and tocompare the midterm results of the twoprostheses we assessed the clinical outcomesof elderly patients with osteoporotic FNFsAOOTA 31BC treated with initial UTAor CTA with a mean followup of yearsMaterials and methodsStudy populationtheandrequirementEthical approval was obtained from theFirst Afï¬liated Hospital of Sun YatsenUniversityforinformed consent was waived by theInvestigational Review Board Consecutiveelderly individuals with the principle diagnosis of an FNF AOOTA 31BC whounderwentinitial UTA or CTA from March to March and forwhom detailed information was availablethroughoutidentiï¬edfrom the orthopaedics department of theFirst Afï¬liated Hospital Sun YatsenUniversity The manufacturer details offollowup were 0cMao et alTable Manufacturer details of stems and cupsemployed in the arthroplasty proceduresProcedureStemCupCORAIL1Exeter3Exeter3REFLECTIONUncemented2UTA n¼ CTA n¼ 1DePuy Synthes Warsaw IN USA2Smith Nephew London UK3Stryker Corporation Kalamazoo MI USAUTA uncemented total hip arthroplasty CTA cementedtotal hip arthroplastyclosed FNFsthe stems and cups employed in the arthroplasty are shown in Table The surgicalprocedure and postoperative rehabilitationprotocol were described in our previouslypublished study11 The inclusion criteriawereAOOTA 31BCactive and cognitively intact patients ageof 15 years independently mobile priorto the injury and a bone mineral densityTscore of at the femoral neck Themajor exclusion criteria were multiple fractures or contralateral limb fractures pathological FNFs lower limb dyskinesia priorto surgery cancer planned surgery polytrauma severe comorbidities eg thyroiddisorder with calcium and phosphorusmetabolism disorderwithcomplications drug abuse affecting bonehealing or bone metabolism early interruption of followup months and cognitive impairment Clinical and radiographicassessments were performed at and months after surgery and every monthsthereafter The primary outcomes were theHarris hip score HHS and the rates ofrevision loosening periprosthetic fractureand dislocationdiabetesStatistical analysisRevision was deï¬ned as partial or completereplacement of the prosthesis12 Looseningof the acetabulum andor stem componentstomographycompared usingas well as dislocation were deï¬ned based ona previous description13 Periprostheticconï¬rmed by Xray orfracture wascomputedexaminationContinuous data ie age bone mineraldensity body mass index followup timeand HHS wereanindependentsamples t test and categoricalvariables ie sex side [leftright] fracturetype comorbidities mechanism of injuryAmerican Society of Anesthesiologists classiï¬cation and major orthopaedic complications were compared using the chisquaretest or the MannWhitney test A KaplanMeier survival curve was used to assess theprobability of revision Hazard ratios werecalculated using a Cox proportional hazards model The signiï¬cance threshold wasset at p The statistical analysis wasperformed using SPSS IBM CorpArmonk NY USAResultsIn total consecutive patientsarthroplasties with an FNF AOOTA31BC who underwentinitial UTA orCTA met our inclusion criteria and wereincluded for analysis Figure The meanfollowup duration was months range months The patients mean age was 06 years for UTA and 06 for CTA The mean body mass indexfor UTA andwas 06 kgm2 for CTA The patientsbaselinesimilarbetween the two groups Table 06 kgm2characteristics werePrimary outcomesImproved functional outcomes were notedin both groups as indicated by the HHSUTA 06 prior to surgery vs 06 at ï¬nal analysis p CTA 06 prior to surgery vs 06 at ï¬nal analysis p At the end of followup the HHS was 0cJournal of International Medical ResearchFigure Flow diagram exhibiting methods for identifying patients with FNFs AOOTA 31BC whounderwent an initial UTA or CTAFNFs femoral neck fractures UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysignificantly different between the twoUTA 06 vs CTAgroups 06 p¼ and patients whounderwent CTA had higherfunctionalscores than those who underwent UTANo distinct betweengroup differenceswere observed at any time point before months postoperatively Table No early year postoperative complications were detectedincluding revisionloosening periprosthetic fracture or dislocation The rate of key orthopaedic complications wasfor UTAand for CTA p Table In the UTA group patients underwent revision UTA developed prosthesis loosening developed periprosthetic fracturesand developed prosthesis dislocation In the CTA group patientsunderwent revision UTA developed prosthesis loosening developed periprosthetic fractures and developed prosthesis dislocation The average time interval from the initial surgery torevision UTA was months range months for UTA and months rangefor CTA p¼ monthsSigniï¬cant differences in revision looseningand periprosthetic fracture were observedbetween the UTA and CTA groups revision vs p¼ loosening 0cMao et alTable Patient demographics and outcomesVariableSex femalemaleAge yearsBMI kgm2BMDSide leftrightFNFs AOOTA type31B31CComorbiditiesDiabetes mellitusHypertensionCerebrovascular diseaseMechanism of FNFsTraffic accidentFallingTamping accidentASA classificationUTA n¼ 06 06 06 06 HHS prior to surgeryData are presented as n n or mean 06 standard deviationpvalueCTA n¼ 06 06 06 06 UTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty HHS Harris hip score ASA AmericanSociety of Anesthesiologists BMI body mass index BMD bone mineral density FNFs femoral neck fracturesTable Comparison of hip functional scoresMonths postoperativelyFinal followupData are presented as mean 06 standard deviationUTA n¼ 06 06 06 06 06 06 06 06 CTA n¼ 06 06 06 06 06 06 06 06 pvalueStatistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty vs p¼ and periprosthetic fracture vs p¼ respectively A significant difference in theprobability of revision was also detectedbetween the groups hazard ratio interval conï¬dencep¼ Figure No significant difference was found in the rate of prosthesis dislocation between the UTA and CTA groups vs respectively 0cJournal of International Medical ResearchTable Rates of key orthopaedic complicationsComplicationsProsthesis revisionProsthesis looseningPeriprosthetic fractureDislocationUTA n¼ CTA n¼ pvalueData are presented as n Statistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplastyFigure KaplanMeier curves showing probability of revision after primary surgery HR was calculatedper the Cox proportional hazards model with age sex American Society of Anesthesiologists classificationbody mass index bone mineral density and femoral neck fracture type as covariates and surgery as the timedependent factorHR hazard ratio CI confidence interval UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysurgeryProbableDiscussionThis review characterised the outcomes of asolitary brand of a total hip arthroplastyimplant during a mean followup of years in elderly patients with osteoporoticFNFs AOOTA 31BC The data demonstrated that patients treated with CTAshowed better improvements in functionaloutcomes and key orthopaedic complications than those treated with UTAThe current ï¬ndings are consistent withprevious studies361415 Although the betterfunctional outcomes and lower rates ofrevision loosening periprosthetic fractureand dislocation are apt to favour CTA nosignificant betweengroup differences in theHHS were detected during the initial yearsafterexplanationsinclude the timedependent clinical efï¬cacyof the implants and the properties of theprostheses34616 Whether UTA or CTA ispreferable in elderly patients with a discontroversial6917placed FNF remainsA recent retrospective study involving patients with an FNF AOOTA 31B whounderwent primary unilateral UTA or CTAshowed that the mean HHS was 06 for CTA and 06 for UTAp¼ A singleblinded randomisedcontrolled trial CHANCEtrial involving individuals treated with an uncementedor cemented tapered hydroxyapatitecoatedfemoral stem and a cemented cup demonstratedtaperedhydroxyapatitecoated femoral stem andcementedthatthe 0cMao et alcemented cup provided better functionalresultsthan the uncemented taperedhydroxyapatitecoated femoral stem andcemented cup16ratesrevisionthe bone microstructureIn the present study the KaplanMeiersurvival curve demonstrated that at the2year analysis neither group showed evidence of a target event and no significantbetweengroup differences were found inofthelooseningfracture or dislocationperiprostheticNeverthelessit would be interesting toexplore whether the prosthesis materialinï¬uencesthepeak effect and the duration of the effectand if so what mechanisms affect the bonemicrostructure and whether there is a wayto change the outcome by blocking thiseffect during a 2year followup We currently have one option for prevention oravoidance of adverse events and thesechanges in treatment strategies may play akey role in improving the clinical resultsif the effects of the prosthesis materialitself cannot be blocked18 There is still alack of consensus on standards for prosthesis revision in this context19When assessing the impact of CTA onthe target events we did not observe anincreased incidence of severe orthopaediccomplications other than the complicationsmentioned in this study In one systematicreview the authors presumed that CTA wassuperior to UTA with respect to functionalscores and tolerable orthopaedic complications20 We obtained analogous results interms of hiprelated complications andfunctional scores Multicentre hip arthroplasty data indicate that UTA remains ahighrisk factor for late revision looseningand periprosthetic fractures810 The notabledissimilarities in the results of these variousstudies on hiprelated complications may belargely attributed to the design of the prosthesis prosthesis size and material selection and the surgeons experience46affected theThis study has several limitations It hada small sample and its retrospective designis association with some inherent disadvantages We did not stratify the patientsaccording to fracture type or sex In addition the potential comorbidities betweengroups were not well exposed and compared The statistical power used to addressdifferences between the groups was insufï¬cient Differences in the patients baselinedata may haveresultsFurthermore our analysis did not determine whether the deaths were instigatedby bone cement The risk of hiprelatedcomplications was not analysed The survivalcurve of other prosthesisrelatedcomplications was estimated using theKaplanMeier method and competitiverisks ie death could have affected thesurvival of the prosthesis Patients whodied lostrevisionHence the revision rate might have beenunderestimated during this long followupwith a fairly high mortality ratethe opportunity forevidenceIn conclusion the ï¬ndings described inthe current review uphold an increasingbody ofthat CTA provideshigher functional scores and lower rates ofhiprelated complications than does UTAin elderly patients with osteoporotic displaced FNFs AOOTA 31BC In thiscontext we recommend CTA for the treatment of such FNFs Our ï¬ndings may beconducive to alleviating continuing debateregarding which prosthesis UTA or CTAis more suitable for the elderly populationA future prospective study may be essentialto conï¬rm whether our conclusion continues to be acceptable as the followup timeincreasesDeclaration of conflicting interestThe authors declare that there is no conï¬ict ofinterest 0cFundingThis research received no speciï¬c grant from anyfunding agency in the public commercial ornotforproï¬t sectorsORCID iDsWeiguang YuGuowei HanReferencesorcid00000001orcid00000003 Chammout G Muren O Laurencikas Eet al More complications with uncementedthan cemented femoral stems in total hipreplacement for displaced femoral neck fractures in the elderly a singleblinded randomized controlled trial with patientsActa Orthop Gjertsen JE Lie SA Fevang JM et al Totalhip replacement after femoral neck fracturesin elderly patients results of fracturesreported to the Norwegian ArthroplastyRegister Acta Orthop Hailer NP Garellick G and Karrholm JUncemented and cemented primary totalhip arthroplastyin the Swedish HipArthroplasty Registerof operations Acta Orthop evaluation Makela KT Eskelinen A Paavolainen Pet al Cementless total hip arthroplasty forprimary osteoarthritis in patients aged years and older results ofthe mostcommon cementless designs compared tocemented reference implants in the FinnishArthroplasty Register Acta Orthop YliKyyny T Sund R Heinanen M et alCemented or uncemented hemiarthroplastyfor the treatment of femoral neck fracturesActa Orthop Yang C Han XL Wang J et al Cementedversus uncemented femoral component totalhip arthroplasty in elderly patients with primary osteoporosisretrospective analysiswith 5year followup J Int Med Res Journal of International Medical Research Solarino G Zagra L Piazzolla A et alceramiconResults of consecutiveceramic cementless hip arthroplastiesinpatients up to years of age a yearsof followup study J Arthroplasty S232S237 Hanly RJ Whitehouse SL Lorimer MFet al The outcome of cemented acetabularcomponents in total hip arthroplasty forosteoarthritis deï¬nes a proï¬ciency thresholdresults of cases from the AustralianOrthopaedic Association NationalJointReplacement Registry J Arthroplasty Imam MA Shehata MSA Elsehili A et alContemporary cemented versus uncementedhemiarthroplasty for the treatment of displaced intracapsular hip fractures a metaanalysis offortytwo thousand fortysixhips Int Orthop Jameson SS Baker PN Mason J et al Thedesign of the acetabular component and sizeof the femoral head inï¬uence the risk of revision following singlebrand cementedhip replacements a retrospective cohortstudy of mediumterm data from a nationaljoint registry J Bone Joint Surg Br 94B Zeng XS Zhan K Zhang LL et alConversion to total hip arthroplasty afterfailed proximal femoral nail antirotationsor dynamic hip screw ï¬xations for stableintertrochanteric femur fractures a retrospective study with a minimum followupof years BMC Musculoskelet Disord Johnson RL Abdel MP Frank RD et alImpact of frailty on outcomes after primaryandarthroplastyJ Arthroplasty 64e5revisiontotalhip Chen KH Tsai SW Wu PK et al Partialcomponentretained twostage reconstruction for chronic infection after uncementedtotal hip arthroplasty results of sixteen casesafter ï¬ve years of followup Int Orthop DeAngelis JP Ademi A Staff I et alCemented versus uncemented hemiarthroplasty for displaced femoral neck fracturesa prospective randomized trial with early 0cMao et alfollowup J Orthop Trauma Rolfson O Donahue GS Hallsten M et alPatientreported outcomes in cemented anduncemented total hip replacements Hip Int Chammout G Muren O Boden H et alCemented compared to uncemented femoralstems in total hip replacement for displacedfemoral neck fractures in the elderly studyprotocol for a singleblinded randomizedCHANCEtrial BMCcontrolled trialMusculoskelet Disord Liu T Hua X Yu W et al Longtermfollowup outcomes for patients undergoingprimary total hip arthroplasty with uncemented versus cemented femoral components a retrospective observational studywith5year minimum followupJ Orthop Surg Res a Engesaeter LB Espehaug B Lie SA et alDoes cement increase the risk of infection inprimary total hip arthroplasty Revisionrates in cemented and uncementedprimary THAs followed for years inthe Norwegian Arthroplasty Register ActaOrthop Schmale GA Lachiewicz PF and Kelley SSEarly failure of revision total hip arthroplasty with cemented precoated femoralcomponents Comparison with uncementedcomponents at to years J Arthroplasty Azegami S Gurusamy KS and Parker MJCemented versus uncemented hemiarthroplasty for hip fractures a systematic reviewof randomised controlled trials Hip Int 0c' | 2 |
"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et | 0 |
" Based on the above analysis we speculated that there may be some interaction between p120ctn isoform 3A and snail which plays a role in suppressing EMT in lung cancer cells expressing cytoplasmic E-cadherin but this hypothesis requires further study. Importantly we also found that knockdown of p120ctn-1A in SPC cells with cytoplasmic E-cadherin resulted in decreased twist expression (B). Meanwhile transfection of LK2 cells which also showed cytoplasmic localization of E-cadherin with the p120ctn isoform 1A plasmid resulted in increased twist expression (B). However no changes in twist expression were observed in the rest of the experiments (A 4A). As a transcription factor and master gene regulator of EMT [39] [40] twist can downregulate E-cadherin expression [41] and upregulate N-cadherin and other mesenchymal biomarkers [42]. Increased twist expression in LK2 cells transfected with the p120ctn isoform 1A plasmid indicated that transcriptional activation took place and further suggested that the p120ctn isoform 1A may have translocated into the nucleus upon binding of E-cad/CTF2 in the cytoplasm consequently activating the Wnt signaling pathway to promote EMT. Decreased twist expression in SPC cells transfected with p120ctn-1A-siRNA indicated that transcriptional activity was downregulated and suggested that ablation of p120ctn isoform 1A resulted in the inhibtion of EMT by removing the stimulatory effect of the Wnt signaling activity by p120ctn isoform 1A. In the H460 and H1299 cells with E-cadherin localized in the membrane the unchanged twist expression confirmed that p120ctn isoforms 1A and 3A could bind to E-cadherin and maintain effective cell-cell adhesion in order to suppress EMT instead of affecting the Wnt/twist pathway. Intriguingly overexpression of p120ctn isoform 3A did not change twist expression in LK2 cells expressing cytoplasmic E-cadherin indicating that p120ctn isoform 3A did not activate transcription. Therefore we firmly believe in the above hypothesis that p120ctn isoform 3A may interact with snail in some manner to influence E-cadherin expression and suppress EMT in lung cancer cells carrying cytoplasmic E-cadherin. Previous studies have observed that p120ctn-1A restored the cytoplasmic expression of E-cadherin whereas p120ctn-3A could not [20] which seems to be contradictory with the results of this study. However the method in previous studies of knocking down p120ctn expression and then transfecting p120ctn isoforms 1A and 3A plasmids into cells is different from that in the current study in which cells were only transiently transfected with p120ctn isoforms 1A and 3A plasmids. Therefore the different research methods may have led to different effects on E-cadherin. We also noted that in previous studies decreased and almost undetectable levels of E-cadherin by ablation of p120ctn resulted in the failure of exogenous p120ctn-1A to translocate into the nucleus to activate the Wnt/b-catenin pathway and decrease E-cadherin expression due to the deletion of the binding partner E-cad/CTF2. However the LK2 and H1299 cell lines used in these experiments expressed E-cadherin in the present study. E-cadherin binds primarily to unphosphorylated p120ctn isoform 3A whereas tyrosine-phosphorylated p120ctn isoform 1A interacts exclusively with N-cadherin [23]. In the previous studies exogenous p120ctn isoform 3A was prevented from binding and stabilizing E-cadherin after its ablation while in the present study the exogenous p120ctn isoform 3A could stabilize E-cadherin expression directly on the membrane or indirectly by increasing its cytoplasmic expression via regulation of snail expression. Of course all of these findings will need to be further investigated. In we for the first time found that p120ctn isoforms 1A and 3A to have different functions in EMT of lung cancer cells with E-cadherin expressed in different subcellular locations. When E-cadherin was localized on the cell membrane p120ctn isoforms 1A and 3A both could inhibit EMT and reduce the cell invasiveness phenotype. When E-cadherin was localized in the cytoplasm p120ctn isoform 1A promoted EMT and enhanced cell invasion while p120ctn isoform 3A inhibited EMT and reduced cell invasiveness. We thank Dr. Albert B. Reynolds (Department of Cancer Biology Vanderbilt University School of Medicine TN USA) for kindly providing p120ctn isoforms 1A and 3A cDNA plasmids. References 1 ThieryJP SleemanJP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. 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Mol Biol Cell20: 2121212919193763 Stat Med Stat Med sim Statistics in Medicine 0277-6715 1097-0258 BlackWell Publishing Ltd Oxford UK 24027094 4098103 10.1002/sim.5963 Research Articles Modeling exposurelagresponse associations with distributed lag non-linear models Gasparrini Antonio * Medical Statistics Department London School of Hygiene and Tropical Medicine London U.K. *Correspondence to: Antonio Gasparrini London School of Hygiene and Tropical Medicine Keppel Street London WC1E 7HT U.K. E-mail: [email protected] 28 2 2014 12 9 2013 33 5 881 899 29 3 2012 10 8 2013 © 2013 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. 2013 This is an open access article under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided the original work is properly cited. In biomedical research a health effect is frequently associated with protracted exposures of varying intensity sustained in the past. The main complexity of modeling and interpreting such phenomena lies in the additional temporal dimension needed to express the association as the risk depends on both intensity and timing of past exposures. This type of dependency is defined here as exposurelagresponse association. In this contribution I illustrate a general statistical framework for such associations established through the extension of distributed lag non-linear models originally developed in time series analysis. This modeling class is based on the definition of a cross-basis obtained by the combination of two functions to flexibly model linear or nonlinear exposure-responses and the lag structure of the relationship respectively. The methodology is illustrated with an example application to cohort data and validated through a simulation study. This modeling framework generalizes to various study designs and regression models and can be applied to study the health effects of protracted exposures to environmental factors drugs or carcinogenic agents among others. © 2013 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. latency distributed lag models exposurelagresponse delayed effects splines 1. Introduction In biomedical research it is commonly appreciated that an exposure event produces effects lasting well beyond the exposure period with an increase in risk occurring from few hours to many years later depending on the physiological processes linking the exposure and the health outcome. The problem is made even more complicated in the presence of protracted time-varying exposures when the health effect measured at a given time can be described as the result of multiple exposure events of different intensities sustained in the past. This phenomenon common to various research fields has been associated for example with peak 1 or chronic exposures 2 to environmental stressors drug intake 34 or occupational exposures to carcinogenic substances 5. The main complexity of modeling and interpreting such dependencies lies in the additional temporal dimension needed to express the association beyond the usual exposureresponse relationship as the risk depends on both intensity and timing of past exposures. Nonetheless the appropriate representation of the temporal pattern of risks may provide further insights on the association of interest in particular regarding the underlying pathophysiological mechanisms and prevent biases in estimates and predictions. Revising previous terminology 6 I define these dependencies as exposurelagresponse associations. In particular this issue has been debated in cancer epidemiology 79. Analytical approaches extend simple indices such as cumulative exposure in order to accommodate the temporal variation in risk because of protracted exposures. In particular the pioneering work by Thomas 106 helped develop sophisticated statistical methods on the basis of weighting past exposures through specific functions whose parameters are estimated by the data. Vacek 11 Langholz and colleagues 12 and Richardson 13 provided interesting applications in case-control studies with weights represented through simple parametric functions. The methodology was improved by Hauptmann and colleagues in a series of papers 1416 by using flexible and smooth spline functions. Sylvestre and Abrahamowicz 17 and Abrahamowicz and colleagues 18 extended the spline methods to the analysis of time-to-event data with a cohort design and presented their applications in pharmaco-epidemiology. The main limitation of the statistical techniques described in these papers is the assumption of a linear exposureresponse relationship. Models for nonlinear dependencies introduce further nontrivial complexities from both statistical and interpretational perspectives as the problem becomes inherently bidimensional. Abrahamowicz and Mackenzie 19 proposed a model for analyzing the nonlinear time-dependent effects of fixed exposures while Vacek 11 and Berhane and colleagues 20 extended this scheme to the case of protracted time-varying exposures. However the modeling techniques illustrated in these other papers still face some limitations as they are based on complex estimation routines with convergence issues and problems in producing uncertainty measures such as standard errors and confidence intervals. Interestingly equivalent approaches were previously established in time series analysis on the basis of distributed lag models (DLMs) a methodology originally formulated in econometrics 21 then applied in epidemiological research 22. These models involve the definition of a distributed lag function analogous to the weighting function described before. In particular Armstrong 23 generalized the method to distributed lag non-linear models (DLNMs) a class of models with different options for the functions applied to model nonlinearity and distributed lag effects. The theory of DLMs and DLNMs have been recently re-evaluated 24 offering a well-grounded statistical tool and a comprehensive scheme for interpretation. In this paper I aim to establish a general conceptual and statistical framework for modeling exposurelagresponse associations built upon the paradigm of DLMs and DLNMs. This modeling class extended beyond time series analysis provides a unified methodology applicable in different study designs data structures and regression models including most of the previous methods as specific cases. Also the statistical framework is defined by completely parametric functions and fitted through standard regression methods with measures of uncertainty and fit statistics easily available. The R package dlnm originally developed for time series data 25 is extended in parallel offering a easy-to-use implementation of the modeling approach. The manuscript is structured as follows. The development and algebraic definition of the modeling framework is described in Section 2. As an illustrative example in I apply the method for investigating the relationship between occupational exposure to radon and lung cancer mortality by using the data from the Colorado Plateau miners cohort. The modeling framework is then validated in a simulation study n. A final discussion is provided in. Information on data and software implementation is included in. The R code and data are included in the supporting information together with additional details making the results of the illustrative example and of the simulation study entirely reproducible. 2. Modeling framework The modeling skeleton is derived by extending the class of DLNMs beyond the time series context. This extension provides a neat algebraic representation and a comprehensive statistical definition. The focus is on a function here defined s(xt) which describes the dependency in terms of the exposure history to x evaluated at time t. The function s(xt) is commonly included in regression models in order to estimate the association while controlling for potential confounders. Although the regression model varies depending on the study design and the type of data the definition of s(xt) provided later and the related modeling framework generally apply. 2.1. Models for linear exposureresponse relationships Previous studies on the topic have defined the function s(xt) by using slightly different algebraic formulae 1026111417. Assuming a linear exposureresponse relationship a general notation can be given by (1a) (1b) (1c) In (1a) the increase in risk at time t is defined as the integral of the instantaneous exposure intensity xu over the period ?t = [t0t1] with t0 and t1 representing the times of the first and last relevant exposures. Here w(t ? u) is the weighting function previously described in which assigns weights to past exposures experienced at time t ? u on the basis of their contribution to the risk at time t. The model can be reparameterized as in (1b) where the risk is now expressed along the lag with ? ? [?0L]. Here L ? ?0 = t1 ? t0 is interpreted as the lag period over which an exposure to x is assumed to affect the risk at time t usually with ?0 = 0. This parameterization offers the advantage that the function w is now directly defined in the new dimension of lag ? and it is independent of the time axis chosen for t which may represent different time scales depending on the study design. The function w(?) termed from here on as the lagresponse function models the lagresponse curve associated with exposure x. Finally for computational purposes the integral is approximated in (1c) by a sum of terms derived by partitioning the lag interval in equally spaced discrete units and assuming the protracted exposure as a sequence of exposure events xt ? ? at lags ? = ?0 ¦ L. A statistical model for (1) can be defined by expressing the lagresponse function w(?) as a linear combination of terms obtained through basis transformation with related parameters. By using matrix notation let the vector qxt of exposure history be defined by (2) Such exposure history changes along time depending on the time t at which the vector qxt is computed. Given (2) the cumulative function s(xt) in (1) can be written using a compact and general matrix notation as (3) The (L ? ?0 + 1) Ã v? matrix C is obtained from the transformation of the lag vector ? = [?0 ¦ ? ¦ L]T by choosing a specific basis with dimension v? for w(?) which defines the related basis functions. In this parameterization the function s(xt) representing the integral of x · w(?) over the interval [?0L] is defined as a lagbasis function with parameters ?. Interestingly the equation in (3) is almost identical to that defining DLMs 24 Eq. (4). The different indexing in the original version reflects the specific application in time series where the data are perfectly ordered in time and the matrix Q has a structure such that qt? ? qt + 1? + 1. However this is a specific case of the general representation in (2)(3). The theory and software already developed for DLMs can be therefore extended in parallel. Alternative lagbasis functions for representing s(xt) are derived through different lagresponse functions w(?) in (1). In particular the traditional index of unweighted cumulative exposure is a specific case of (3) where reduces to with w(?) equal to a constant c. This is obtained by specifying C as an (L ? ?0 + 1)-dimensional vector of 1's with v? = 1." | 1 |
Molecular Medicine The role of selenium metabolism andselenoproteins in cartilage homeostasisand arthropathiesDonghyun Kang Jeeyeon Lee Cuiyan Wu3 Xiong Guo3 Byeong Jae Lee24 JangSoo Chun5 andJinHong Kim AbstractAs an essential nutrient and trace element selenium is required for living anisms and its beneï¬cial roles in humanhealth have been well recognized The role of selenium is mainly played through selenoproteins synthesized by theselenium metabolic system Selenoproteins have a wide range of cellular functions including regulation of seleniumtransport thyroid hormones immunity and redox homeostasis Selenium deï¬ciency contributes to various diseasessuch as cardiovascular disease cancer liver disease and arthropathyKashinBeck disease KBD and osteoarthritisOA A skeletal developmental disorder KBD has been reported in lowselenium areas of China North Korea and theSiberian region of Russia and can be alleviated by selenium supplementation OA the most common form of arthritisis a degenerative disease caused by an imbalance in matrix metabolism and is characterized by cartilage destructionOxidative stress serves as a major cause of the initiation of OA pathogenesis Selenium deï¬ciency and dysregulation ofselenoproteins are associated with impairments to redox homeostasis in cartilage We review the recently exploredroles of selenium metabolism and selenoproteins in cartilage with an emphasis on two arthropathies KBD and OAMoreover we discuss the potential of therapeutic strategies targeting the biological functions of selenium andselenoproteins for OA treatmentIntroductionSelenium Se is an essential trace element in humans12Selenium is generally taken up from the diet through foodor other forms of external supplementation Dietaryselenium is obtained in the form of selenomethionineSeMet selenocysteine Sec selenite and selenate Signiï¬cant health beneï¬ts have been attributed to seleniummetabolic systems that play major physiological roles inthyroid hormone metabolism immunity and antioxidantdefense23 Selenium is required for the production ofthyroid hormonemetabolizing enzymes and seleniumCorrespondence JinHong Kim jinhkimsnuackr1Center for RNA Research Institute for Basic Science Seoul South Korea2Department of Biological Sciences College of Natural Sciences Seoul NationalUniversity Seoul South KoreaFull list of author information is available at the end of the These authors contributed equally Donghyun Kang Jeeyeon Leesupplementation is thought to improve the function ofthyrocytes and immune cells4 Selenium supplementationdemonstrated immunostimulant effects such as enhancedproliferation of activated T cells activation of naturalkiller cells and tumor cytotoxicity mediated by cytotoxiclymphocytes56 In contrast selenium deï¬ciency is associated with the occurrence virulence and disease progression of viral infections7Selenium inadequacy can lead to various types ofdiseases most notably cardiovascular disease8 cancer13 hepatopathy1617 and arthropathy Cardiovascular diseases are associated with systemic seleniumlevel with a higher risk at or μgL seleniumconcentration in the blood10 A type of endemic cardiomyopathy Keshan disease is linked to selenium deï¬ciency811 Keshan disease occurs in lowselenium areasin Chinasodium seleniteand is prevented by The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Korean Society for Biochemistry and Molecular Biology 0cKang Experimental Molecular MedicinePage of studiesEpidemiologicalsupplementation12 Lowselenium status is correlatedwith a significantly increased risk of cancer incidenceand mortality13haveprovided evidence on the cancerpreventing effects ofselenium18 Selenium deï¬ciency is also characterizedby elevated levels of oxidative stress markers in the liver21which significantly contribute to liver injury17 The oxidative stress caused by selenium deï¬ciency further plays adetrimental role in joint development Selenium deï¬ciency is the main cause of endemic KashinBeck diseaseKBD which is mainly reported in lowselenium areas ofChina North Korea and the Siberian region of RussiaMoreover there is a growing body of evidence suggestingthat the pathogenesis of osteoarthritis OA the mostcommon form of arthritis may be associated with selenium deï¬ciency by resulting in oxidative stress22However it is noteworthy that excessive selenium intakecan also cause selenosis2930 which accompanies adversesymptoms including fatigue diarrhea nausea increasedheart rate necrosis in liver and kidney and neurologicaldamage Chroniccompromisesimmune and reproductive systems in humanseventuallyselenosisOA is characterized by progressive loss of cartilageextracellular matrix ECM and pathological changes inother joint tissues such as subchondral bone sclerosisosteophyte formation and synovial ammation31 Cartilage destruction is considered a hallmark of OA and is aresult of increased production of catabolic effectors32and reduced matrix biosynthesis by chondrocytes36 OA isassociated with multiple etiologies involving systemicfactors such as age37 as well as local factors such asmechanical stress38 driven by weightbearing and jointinstability Both OAcausing factors have been found tocause oxidative stress in chondrocytes Oxidative stressresults from the abnormal production of reactive oxygenspecies ROS and the loss of cellular antioxidant capacityMany preclinical and clinical studies have indicated theaccumulation of oxidative burden in chondrocytesundergoing osteoarthritic changes3940 Emerging evidence suggests that oxidative stress is mechanisticallylinked to the initiation of osteoarthritic changes inchondrocytes through the acquisition of senescent phenotypes36 Therefore restoring redox homeostasis canserve as a rational therapeutic strategy to alleviate OAprogression Here we review the role of selenium metabolism in cartilage and bone and the signiï¬cance ofmaintaining its homeostasis in the context of joint diseases such as KBD and OAOverview of the selenium metabolic systemThe selenium metabolic system and the biosynthesis ofselenoproteinsSelenium metabolism is a systemic process that includesandtransformationtransportationabsorptiontheOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyexcretion of selenium Fig Selenium is obtained inanic formsSeMet and Secand inanic formsselenite and selenatefrom diet Selenium is taken up bythe liver that synthesizes and exports SELENOP whicheventually circulates through the bloodstream SELENOPwith multiple Sec residues41 transports selenium to othertissues and ans42 and the transported selenium isconverted to selenophosphate by intracellular seleniummetabolic pathways Selenium is excreted through exhalation and urine in the form of smallmolecule metabolites formed by sequential methylation4344Selenium plays biological roles predominantly in theform of selenoproteins synthesized by the seleniummetabolic system Ingested inanic selenium is ï¬rstreduced to hydrogen selenide H2Se via glutathioneGSH and thioredoxin TXN systems Selenide is furtherconverted to Sec amino acids for incorporation intospeciï¬c sites of selenoproteins such as the catalytic sites ofa selenoenzyme Mechanistically selenophosphate synthetase SEPHS2 catalyzes the production of selenophosphate through the reduction of hydrogen selenideThe subsequent reaction with phosphoseryltRNA PSertRNA[Ser]Sec yields SectRNA[Ser]Sec Sec amino acids areincorporated into polypeptidethrough themachinery utilizing the UGA codon Selenocysteineinsertion sequence binding protein SBP2 binds toselenocysteine insertion sequence SECIS element whichis located in the ²untranslated region ²UTR of selenoprotein mRNA and mediates the transfer of SectRNA[Ser]Sec to the Asite of ribosome which recognizesthe UGA codon as the Sec integration codon Collectivelythe selenoprotein translation machinery consists of SECISelement SBP2 Secspeciï¬c eukaryotic elongation factorEEFSEC and aminoacylated SectRNA[Ser]Sec therebyenabling UGA to be recognized as a Sec codon and utilized for translation into the growing polypeptidechainsSelenoproteinssome ofSelenoprotein is deï¬ned as a protein containing Secamino acid residue The biological functions of seleniumare mostly exerted through selenoprotein domains thatcontain Sec residues Twentyï¬ve selenoprotein geneshave been identiï¬ed in the human genome45 In mice atotal of selenoproteins have been characterized46 andtargeted deletion ofthese selenoproteinsdemonstrated their essential roles in developmental processes and in disease pathogenesis Selenoproteins can beclassiï¬ed into subfamilies based on their cellular functionssuch as those implicated in antioxidation GPX1 GPX2GPX3 GPX4 redox regulation TXNRD1 TXNRD2TXNRD3 MSRB1 SELENOH SELENOM SELENOWthyroid hormone metabolism DIO1 DIO2 DIO3 selenium transport and storage SELENOP selenophosphatesynthesis SEPHS2 calcium metabolism SELENOK 0cKang Experimental Molecular MedicinePage of Fig Selenium metabolic system in mammals Selenium is absorbed from the diet undergoes several conversion steps and is incorporated intopolypeptide chains completing selenoprotein synthesis Dietary sources of selenium uptake exist in inanic form such as selenate and selenite andanic form such as Sec and SeMet Inanic forms are reduced by TXNRDTRX or GRXGSH systems and anic forms are cleaved by SCLYforming selenide Selenophosphate is synthesized from selenide by SEPHS2 and the subsequent reaction with PSertRNA[Ser]Sec mediated by SEPSECSyields SectRNA[Ser]Sec SectRNA[Ser]Sec is transferred to the Asite of ribosome mediated by SBP2 which binds to SECIS located in the ²UTR of aselenoprotein mRNA Finally the UGA codon is recognized as the Sec integration codon Abbreviations SeMet selenomethionine Secselenocysteine GRX glutathione reductase TRX thioredoxin TXNRD thioredoxin reductase GSH glutathione MGL methionine gammalyase SCLYselenocysteine lyase SEPHS2 selenophosphate synthetase SARS seryltRNA synthetase PSTK phosphoserylSeptRNA kinase SEPSECS SeptRNASectRNA synthase EEFSEC Secspeciï¬c eukaryotic elongation factor SBP2 SECIS binding protein SELENOT myogenesis SELENON protein foldingSELENOF SELENOI SELENOS and protein AMPylation SELENOO4748 The functions of other selenoproteins such as GPX6 and SELENOV still remain unclearGlutathione peroxidases GPXs such as GPX1 cytosolicGPX GPX2 gastrointestinal GPX and GPX4 phospholipid hydroperoxide GPX catalyze the decompositionof a great variety of peroxides thus protecting cellsagainst oxidative damage4950 Thioredoxin reductasesTXNRDs employ NADPH as an electron donor to revertoxidized TXN to a reduced dithiol the oxidation status ofwhich is critically implicated in regulating various cellbehaviors including proliferation and apoptosis51 Thephysiological signiï¬cance of TXNRDs is further supported by the embryonic lethality of Txnrd1 or Txnrd2knockout mice5253 Deiodinases DIOs regulate thyroidhormone metabolism by catalyzing the conversion ofthyroid hormones from precursor thyroxine T4 to biologically active triiodothyronine T3 or inactive reverseT3 rT354 The expression levels of several selenoproteinsOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyare uenced by the extent of selenium uptake Forexample seleniumdeï¬cient animals and human cell linesexhibit reduced transcription of selenoproteins such asGPX1 DIOs SELENOI and SELENOW55 A subset ofselenoproteins such as GPX1 and SELENOW is moresensitive to selenium supplementation or deï¬ciency Thehierarchy of selenoprotein expression is more apparentwhen the intracellular level of selenium is limited1Seleniumresponsive genesgenesareseleniumcontainingSeleniumresponsivethe genes whoseexpression patterns are uenced by supplementationwith selenium orcompoundsTreatment of a cancer cell line with methylseleninic acidin genes58 Theseinduced expression changesresponsive genes were closely associated with annotationsrelated to cell cycle regulation androgenresponsive genesand phase II detoxiï¬cation pathway Selenium supplementation of macrophages diminished the expression oflipopolysaccharide LPSinduced proammatory genes 0cKang Experimental Molecular MedicinePage of such as cyclooxygenase2 COX2 and tumor necrosisfactorα TNFα59 suggesting that selenium has antiammatory effects on the immune system The CTDdatabase httpctdbase reports the effect of environmental chemicals including selenium on gene expression proï¬les in various human tissuesThe role of selenium and selenoproteins incartilage development and KBDSelenium levels and its role in joint tissuesJoints are composed of various types of connective tissues including cartilage bone synovium meniscus andligament Among these tissues cartilage is the maincomponent that absorbs mechanical stress cushioningbones from impacting each other during various weightbearing activities In the human knee joint the seleniumconcentration in cartilage is approximately μgkg dryweight whereas the selenium concentrations in ligamentand meniscus are and μgkg dry weight respectively6061 The requirement of adequate physiologicalselenium levels for maintaining cartilage homeostasis hasbeen recognized Selenium deï¬ciency retards the growthand development of cartilage and bone62 Growthretardation was observed in rats after two generations ofselenium deï¬ciency62 Mice fed a diet deï¬cient in selenium resulted in ï¬brocartilage formation at the articularsurface ultimately showing degeneration of articularcartilage63 Selenium deï¬ciency induced the expression ofthe chondrocyte hypertrophy marker gene type X collagenCOLX in articular cartilage64 The expression of parathyroid hormonerelated protein PTHrP which controlschondrocyte maturation during endochondral ossiï¬cation was enhanced in both articular cartilage andhypertrophic growth plate following selenium deï¬ciencyThese changes were in line with the phenotypic changesobserved in the cartilage of KBD patients64 However itshould be noted that growth retardation caused by selenium deï¬ciency may also be associated with the deregulation of bone metabolism65 In a study by Cao et alselenium deï¬ciency severely compromised bone microarchitecture as a result of increased bone resorption66Abnormalities in selenium metabolism and skeletaldevelopment diseasesSelenium deï¬ciency is regarded as one of the initiatingfactors of KBD which is an endemic osteoarthropathycaused by the premature closure of epiphyseal plate andthe impaired skeletal development Skeletal deformities inhands ï¬ngers knees and elbows and in severe casesdwarï¬sm and movement disorders are the symptoms ofKBD22 The KBD area roughly coincides with lowselenium areas including a geological belt extendingfrom northeast to southwest China North Korea andeastern Siberia22 A metaanalysis showed that seleniumOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologylevels in the water soil cereal and corn in KBD endemicregions were lower than they were in nonendemicregions supporting the fact that the level of selenium intissue is predominantly affected by dietary intake23 In linewith this ï¬nding selenium levels in the whole bloodserum hair and urine of KBD patients were markedlylower than those of healthy controls24Selenoprotein gene polymorphisms are associated withincreased susceptibility to KBD There were significantdifferences in the allelic frequency of GPX1 Pro198Leurs1050450 between the KBD and control group67 Inaddition the mRNA level of GPX1 and enzyme activity oftotal GPX in blood were lower in the KBD group thanthey were in the control group67 Haplotypes of TCCTTC and TTT of rs1050450 rs3811699 and rs1800668in GPX1 gene also had a significant link to KBD68 Asinglenucleotide polymorphism SNP in the promoterregion of SELENOS rs28665122 105G A was relatedto the increased risk of KBD and upregulation of PI3KAktsignaling in patients with KBD69 In this study tertbutylhydroperoxide tBHPtreatmentinduced chondrocyteapoptosis was mitigated by selenium supplementation viasodium selenitetreatment which suppressed thePI3KAkt pathway The minor Aallele of SELENOFrs5859 was associated with a significantly higher incidenceof KBD70The animals fed a seleniumdeï¬cient diet recapitulatedsome of the pathological manifestations of KBD stronglysupporting the notion that selenium deï¬ciency is criticallyassociated with the development of this endemic arthropathy Selenium deï¬ciency impaired bone and cartilagegrowth with the exhibition of premature chondrocytehypertrophy as evidenced by an increased expression ofCOLX compatible with the phenotypes in KBD cartilage64The lowselenium condition in combination with threemycotoxins deoxynivalenol DON nivalenol NIV and T yielded procatabolic changes and hypertrophic phenotype of chondrocytes as evidenced by the loss of aggrecanand type II collagen COLII and the increase in COLX andmatrix metalloproteinases MMPs expressionrespectively71 In contrast selenium supplementation partiallyalleviated these mycotoxininduced damages in chondrocytes71 In rats dietary selenium deï¬ciency over twogenerations caused the onset of physiological seleniuminsufï¬ciency72 In this condition pathological changes inthe epiphyseal plate were observed with the decreasedexpression of COLII and GPX1 in the chondrocytes suggesting a possible association of reduced chondrocyte anabolism and antioxidant capacity with the epiphyseal platelesions observed in KBD72 The relevance ofimpairedselenium metabolism to the onset of KBD was furthervalidated using a mouse genetic deletion model Targeteddeletion of SectRNA[Ser]Sec Trsp gene in osteochondroprogenitor cells from embryonic stage caused the 0cKang Experimental Molecular MedicinePage of depletion of selenoproteins in skeletal systems causinggrowth retardation abnormalities in the epiphyseal growthplate delayed endochondral ossiï¬cationand chondronecrosis which recapitulated the major pathologicalfeatures of KBD73As a prophylactic treatment selenium supplementationswere given to children living in a KBD area The supplemented group showed elevated physiological seleniumlevels in their hair samples and exhibited a substantiallylower prevalence of KBD74 A metaanalysis including ï¬verandomized controlled trials RCTs and ten prospectivenonRCTs statistically demonstrated the beneï¬ts of selenium supplementation in preventing KBD in children75Selenium metabolism and OAPhysiological signiï¬cance of oxidative stress inchondrocytesOA is the most common form of arthritis and is primarilycharacterized by the loss of cartilagespeciï¬c ECM and otherpathological changes in joints including subchondral bonesclerosis osteophyte formation and synovial ammation31Articular cartilage is composed of abundant proteoglycans inwhich sulfated glycosaminoglycan chains such as chondroitinsulfates are bound to a core protein such as aggrecan Loss ofcartilage matrix during OA progression is a combined resultof increased catabolic process in cartilage and reduced anabolic activity of chondrocytes The molecularlevel understanding of OA pathogenesis has led to the identiï¬cation ofmajor catabolic enzymes ADAMTS576 MMP377 andMMP1378 which mediate the degradation of cartilagematrix Proammatory cytokines drive the expression ofthese catabolic factors in chondrocytes through the activationof transcription factors such as HIF2α32 and NFκB79Abnormalities in various metabolic pathways such as glucose80 or amino acid metabolic system81 in chondrocyteshave been implicated in activating catabolic cascades inosteoarthritic cartilage82 Moreover increased cellular uptakeof Zn2 through the upregulation of zinc transporter ZIP8activates metalregulatory transcription factor1 MTF1which in turn induces the expression of matrixdegradingenzymes in chondrocytes3383 Regulation of catabolism bythefurthershowed the association of metabolic abnormalities with thecatabolic process of OA34cholesterolCH25HCYP7B1RORαaxisMeanwhile the upstream regulatory mechanism eliciting an imbalance in OA matrix homeostasis needs furtherinvestigation OAcausing factorssuch as age andmechanical stress lead to excessive oxidative stress inchondrocytes3738 Consistently clinical and preclinicalOA studies indicated a cumulative oxidative burden inosteoarthritic chondrocytes3940 Emerging evidence suggests that oxidative stress plays a significant role in OAdevelopment and the disease progression can be mitigatedby counteracting oxidative stress3684In generalOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyoxidative stress results from the abnormal production ofROS and the loss of cellular antioxidant capacity Synovialï¬uid from patients with latestage OA who were undergoing knee joint replacement had a lower level of oxidoreductases than that from healthy controls87 In partthe increase in oxidative stress is attributable to mitochondrial dysfunction in OA chondrocytes8889 OAchondrocytes displayed reduced mitochondrial DNAcontent mitochondrial dysfunction and diminishedexpression of NRF2 which regulates the transcription ofoxidoreductase genes89 Similarly chondrocytes fromaged individuals exhibited increased ROS burden andmitochondrial and genomic DNA damage90 Therefore the proper maintenance of redox homeostasis canpotentially serve as a rational therapeutic strategy toprotect against OA progressionPotential roles of selenium metabolism in OAThe protective effect of selenium in OA has beenexplored in a large number of epidemiological and geneticstudies Table The concentration of selenium in serumwas significantly lower in OA patients than that of normalcontrols25 Similarly the results from a populationbasedcohort study demonstrated the linkage between lowselenium levels in toenails with OAassociated pain anddisease severity2627 Several studies have indicated thatcartilage matrix homeostasis is impaired in seleniumdeï¬ciency Lowselenium status diminished COLIIexpression level regulated by SOX9 which is known as amaster regulator required for maintaining cartilage matrixIn fact SOX9 was destabilized by thehomeostasisdownregulation ofseleniumresponsive PRMT5 thatsustains SOX9 stability via methylation93 In anotherstudy rats fed a seleniumdeï¬cient diet exhibited lowsulfotransferase activity which resulted in diminishedforcontents ofmechanicalcartilagematrix28 In contrast selenium supplementation ameliorated the spontaneous degeneration of articular cartilagein STR1 N mice by increasing the expression of GPXs94In cultured chondrocytes pretreatment with SeMetmarkedly inhibited nitric oxide NO and prostaglandinE2 PGE2 production in response to proammatorycytokine IL1β95 Expression of SBP2 a factor recognizingSECIS element had a positive correlation with GPX1 andGPX4 expression and antioxidant capacity in chondrocytes96 Oxidation resistance mediated by SBP2 wasdiminished in response to IL1β treatment in vitro and indamaged regions of cartilage in OA patients96 Downregulation of selenoprotein mRNAs including GPX397GPX1 and GPX49698 and Selenop99 was observed inhuman and mouse OA chondrocytessulfated glycosaminoglycan essentialstressabsorbingpropertyofGenetic factors such as SNPs in selenoproteins wereidentiï¬ed to be risk factors for OA development A GAG 0cKang Experimental Molecular MedicinePage of Table List of selenoproteins associated with the pathogenesis of arthropathies KBD and OAGeneGPX1GPX3GPX4DIO2DIO3SELENOFSELENOPSELENOSFunctionExpression in OASNPAntioxidantReduction of hydrogen peroxide and anic peroxidesDownregulatedPlasma antioxidantDetoxiï¬cation of lipid hydroperoxidesMetabolism of lipidsActivation of hormonesDeiodination of T4 to T3Inactivation of hormonesConversion of T4 to rT3Protein foldingStorage and transport of SeAntioxidant propertiesProtein foldingERassociated protein degradationDownregulatedDownregulatedUpregulatedDownregulatedrs1050450 KBDrs3811699 KBDrs1800668 KBDrs225014 OArs12885300 OArs945006 OArs5859 KBDrs28665122 KBDRefhaplotype in SELENOS gene was significantly associatedwith increased levels ofammatory factors in OApatient plasma100 SNPs in DIO2 which converts precursor thyroid hormone T4 to its active form T3 were alsorelated to genetic susceptibility to OA developmentLevels of DIO2 mRNA and protein were markedly upregulated in OA cartilage101 A common DIO2 haplotypecomposed of the minor Callele of SNP rs225014 and thecommon Callele of SNP rs12885300 was significantlyassociated with advanced hip OA as indicated by a higherodds ratio101 Locus rs225014 which confers risk toOA was associated with the differential methylation ofCpG located in the upstream region of DIO2 gene andwas correlated with upregulated DIO2 expression inOA104 Meanwhile DIO3 depletes the resources that canbe utilized for the production of active thyroid moleculesby catalyzing the conversion of T4 and T3 into inactivemetabolites The minor Gallele of the DIO3 variantrs945006 was associated with a protective effect againstOA development105However a few aspects regarding the relationshipbetween selenium and OA remain controversial Firstseveral studies indicate that there are no differences inselenium levels between OA and normal tissues Theselenium concentrations in synovial ï¬uid and plasma of OA patients were not significantly different from thoseof healthy controls106 Similarly no significant difference in selenium concentration was noted between sixdogs with posttraumatic OA and six control dogs107Second the beneï¬cial effect of selenium supplementationin alleviating OA symptoms has been debated The resultsfrom a controlled doubleblind trial of patientsOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyrevealed that the supplementation of a formulation containing selenium with vitamins A C and E SeACE didnot have any remarkable curative effect compared to aplacebo108 In a study with an independent cohort theprevalence of radiographic knee OA was not significantlyassociated with dietary selenium intake109Nonethelessit is apparent that selenium deï¬ciencydysregulation of selenoproteins and genetic variations inselenoprotein genes serve as potential risk factors for OAThe vital role of selenium metabolism in maintainingcartilage homeostasis is expected considering its criticalinvolvementin regulating cellular processes such aschondrogenic differentiation of progenitor cells maintenance of redox homeostasis and DNA damage repair inchondrocytes which are covered in the next sectionIntracellular roles of selenium metabolism andselenoproteins in cartilageChondrogenic differentiation programs of progenitor cellsSelenium exerts various beneï¬cial effects to promoteproliferation and differentiation of chondrogenic progenitorcells110111 Selenium supplementation stimulated the proliferation of ATDC5 chondrogenic cells even under serumdeprivation by inducing cyclin D1 expression110 Deï¬ciencyof SELENOO interfered with the chondrogenic differentiation of ATDC5 cells by suppressing the expression ofchondrogenic genes SOX9 COLII and aggrecan anddecreasing the activity of alkaline phosphatase112 Knockdown of Gpx1 reduced the chondrogenic differentiation ofATDC5 cells by modulating intracellular GSHoxidizedGSH GSSG ratio113 Selenop was differentially upregulatedduring the chondrogenic differentiation of micromass 0cKang Experimental Molecular MedicinePage of culture of mesenchymal cells isolated from mouse limbbuds114 In line with the effects of selenium metabolism andselenoproteins in chondrogenic progenitor cells observedin vitro deï¬cient uptake of selenium severely affectedchondrogenic differentiation of mesenchymal lineage cellsin mice64andOsteochondroprogenitorspeciï¬c deletion of Trsp genesignificantly impaired chondrogenic programs causingabnormalities in bone and cartilage development in mice73endochondralossiï¬cationthusAntioxidant defense and redox homeostasisfunction ofattributed to theThe protective effects of selenium on cartilage are primarilyantioxidantdefense115 The metabolism and survival of chondrogenic progenitors and chondrocytes are greatly compromised by ROS including free radicals peroxides andsuperoxide anions118 Recent studies strongly supportthe notion that mitochondrial dysfunction and oxidativestress are the main drivers of OA pathogenesis37Although ROS play essential roles in the maintenance ofbasal cellular activities such as chondrocyte proliferationand matrix remodeling in cartilage excessive oxidativestress causes detrimental events such as cellular senescence36121 dedifferentiation122 and apoptosis123 ROScause oxidative damage to various cellular componentsand disrupt the balance between ECM catabolism andanabolism119 ROS suppress mitochondrial oxidativephosphorylation and ATP production which are essentially required to sustain cartilage matrix synthesis124 Inaddition ROS induce matrix degeneration through theupregulation of matrixdegrading enzyme expressionwhile this effecttreatment123125 The detrimental effects of ROS on cartilagehomeostasis can be effectively alleviated by augmentingcellular antioxidant activity under stress conditions andseveral attempts have been made to treat OA by targetingthe regulators involved in oxidative stress production incartilage84is abolished by antioxidantThe protective role of selenium metabolism is thoughtto be exerted through the neutralization of ROS viaantioxidant activities of selenoproteins including GPXsand TXNRDs Bone marrow stromal cells cultured inmedium supplemented with low selenite concentrationexhibited ROS accumulation along with the reducedexpression of GPXs TXNRDs and other seleniumindependentinmicronuclei generation which is an indication of chromosome damage126 Both GPX1 expression and activitywere substantially lower in mice fed a seleniumdeï¬cientdiet than those in mice fed a normal dietleading todecreased trabecular number reduced femoral trabecularvolumetotal bone volume ratio and trabecular separation66 The rats exposed to a seleniumdeï¬cient diet withT2 toxin showed increased lipid peroxidation level andoxidoreductaseenzymesresultingOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologydecreased antioxidant GPX activity in their serum andcartilage127 A seleniumdeï¬cient dietinduced theexpression of miR1385p which in turn suppressed theexpression of SELENOM that has antioxidant functionand caused mitochondrial dysfunction and apoptosis ofchondrocytes128 Lead Pbinduced oxidative stress andtoxicity reduced the expression of selenoprotein mRNAsand the effect was mitigated by selenium supplementation129 In summary the antioxidant properties of selenoproteins showed therapeutic potential by counteractingthe accumulation of damage induced by oxidative stress incartilageDNA damage repairIt is well known that DNA damage pathways play substantial roles in the progression of arthropathies119 Theexpression of genes related to DNA damage was changedin the cartilage of KBD patients130131 Chronic DNAdamage induces the initiation of apoptosis or cellularsenescence in chondrocytes36132133 Selenium has apotential to reduce DNA damage and increase DNArepair capacity134 In part the beneï¬cial effect of seleniumon genomic stability is associated with the antioxidationeffect of selenoproteins such as GPXs and TXNRDswhich remove ROS before they cause DNA damage134Cancer cells supplemented with selenium nM sodiumselenite or μM SeMet showed elevated levels of GPX1and TXNRD1 enzyme activity effectively protectingagainst DNA strand breaks induced by ultraviolet A orH2O2induced oxidative stress135 SeMet reduced theextent of DNA damage and enhanced DNA repair capacity by inducing repair complex formation in DNAdamaged cells through U | 2 |
" inflammatory bowel disease ibd is the collective term for chronic immunemediated diseases ofunknown multifactorial etiology arising from the interplay between genetic and environmental factors andincluding two main disease manifestations ulcerative colitis uc and crohns disease in the last few decadesnaturally occurring alkaloids have gained interest because of their substantial antiinflammatory effects in severalanimal models of disease studies on mouse models of ibd have demonstrated the antiinflammatory action of themain tobacco alkaloid nicotine in addition anatabine a minor tobacco alkaloid also present in peppers tomatoand eggplant presents antiinflammatory properties in vivo and in vitro in this study we aimed to evaluate theantiinflammatory properties of nicotine and anatabine in a dextran sulfate sodium dss mouse model of ucresults oral administration of anatabine but not nicotine reduced the clinical symptoms of dssinduced colitisthe result of gene expression analysis suggested that anatabine had a restorative effect on global dssinducedgene expression profiles while nicotine only had limited effects accordingly map findings revealed that anatabinereduced the colonic abundance of dssassociated cytokines and increased il10 abundances our results support the amelioration of inflammatory effects by anatabine in the dss mouse modelof uc and suggest that anatabine constitutes a promising therapeutic agent for ibd treatmentkeywords plantderived alkaloid mouse model nicotine colitis correspondence pedroantonioruizcastropmicomblainephillipspmicom juliahoengpmicom pedro a ruiz castro ulrike kogel giuseppe lo sasso blaine w phillips andalain sewer contributed equally to this work1philip morris international rd philip morris products sa quai jeanrenaud neuch¢tel switzerland2philip morris international research laboratories pte ltd science parkroad the kendall science park ii singapore singapore the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cruiz castro of inflammation page of crohns disease cd and ulcerative colitis uc the mainclinical phenotypes of inflammatory bowel diseases ibdare chronic relapsing inflammatory disorders that affectthe gastrointestinal tract ibd are thought to result froman inappropriate and continuing inflammatory responseto commensal microbes in a genetically susceptible hostenvironmental triggers such as increased hygiene druguse stress smoking and diet influence the onset of thedisease over the past decades naturally occurring alkaloids from plant or medicinal herb sources have sparkedconsiderable interest because of their significant antiinflammatory and antioxidant properties []alkaloidsa class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atomsare produced by a widerange of living anisms such as bacteria fungi plantsand animals in plants alkaloids are produced assecondary metabolites in response to environmentalbiotic or abiotic interactions and they confer protectionthrough a range of insecticidal antimicrobial and pharmacological attributes the antiinflammatory activities ofplantderived alkaloids have been documented in severalanimal models of disease including respiratory distress spinal cord injury hepatic fibrosis cancer and ibd [ ] the protective effects of alkaloids havebeen attributed to amelioration of inflammatory responsesand colonic oxidative stress [ ] promotion of epithelial barrier function and positive regulation of gutmicrobiota pyridine alkaloids present in tobacco nicotiana tabacum have been the subject of intensive research nicotinethe major alkaloid in tobacco accounts for approximately of the total alkaloid content of tobacco while thestructurally related nornicotine and anatabine are themost abundant minor pyridine alkaloids accounting for to of total alkaloids other pyridine alkaloids intobacco such as anabasine anabaseine and cotinine arepresent in smaller amounts nicotine and all minortobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholinereceptors nachrs tobacco nachr agonists suchas nicotine anatabine anabasine anabaseine and cotininedisplay protective effects in animal models of several inflammatory conditions including sepsis parkinsonsdisease alzheimers disease and ibd several in vitro and in vivo studies have shown a clearnicotinedependent positive effect on inflammatory processes [ ] in a previous study oral nicotine administration reduced the severity of dssinduced colitis andreduced colonic tnfα synthesis while subcutaneous injection or minipump infusion had no effect highlightingthe crucial role of administration route for the protectiveeffects of nicotine in dss colitis nicotine has alsobeen shown to attenuate the severity of dss colitis andexpression of il6 in cd4t cells a recent studysuggested that nicotine ameliorates dssinduced inflammation through inhibition of signaltransducer andactivator oftranscription stat3 in gutinfiltratedlymphocytes and intestinal epithelial cells recentlynicotine was shown to cause a decrease in leukocyte recruitment disease activity index dai and histologicalscore in dss colitis and block tnfmediated expressionof mucosal vascular addresin cell adhesion molecule1 inendothelial cells these authors concluded that nicotinesuppressesinflammation through downregulation ofadhesion molecules in the gut nonetheless clinicalstudies on the efficacy and tolerability of nicotine haveshown thattherapeutic application of nicotine fortreatment of uc is limited because of frequent adverseeffects and nicotine inconsistent efficacy in maintainingremission in uc patients [ ]anatabine is found in plants of the solanacea familywhich includes tobacco peppers tomato and eggplant little is known about the biological properties of anatabinealthough several studies have suggested that this alkaloid is apotential candidate compound for antiinflammatory drugdevelopment [ ] anatabine was marketed in the us asa dietary supplement under the name anatabloc in aninternetbased survey approximately of all users ratedthe effect of anatabine supplementation as good or excellentfor joint pain stiffness and functionality anatabine hasbeen shown to inhibit lipopolysaccharide lpsinducedproinflammatory gene expression as well as nfκb andstat3 phosphorylation in human neuroblastoma shsy5y hek293 human microglia and human bloodmononuclear cells as well as in the brain and spleen ofmouse models of autoimmune encephalomyelitis andalzheimers disease in shsy5y cells anatabine alsoreduced the expression of betasecretase the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimers diseasethrough inhibition of nfκb activation in this study we aimed to assess the antiinflammatoryeffects of the tobacco alkaloids nicotine and anatabine inthe established dss mouse model of uc our resultsshow that oral administration of anatabine but notnicotine ameliorates the clinical manifestations of dsstreatment in mice the results of gene expression analysisindicated that anatabine had a partial restorative effect onglobal dssinduced gene expression profiles while nicotineonly had minimal effects moreover multianalyte profilingmap showed that anatabine but not nicotine suppressesthe production of il6 il1α tnfα granulocytecolonystimulating factor gcsf and keratinocyte chemoattractant kc while increasing il10 expression in the colon ofdsstreated mice for an overview of the study conceptand analytical procedures see online resource 0cruiz castro of inflammation page of resultsanatabine has a protective effect in the dss mousemodel of colitisto study the antiinflammatory properties of nicotineand anatabine c57bl6 mice were provided with nicotine or anatabine in drinking water for a total of dayscolitis was induced by oral administration of dssin drinking water ad libitum during days fig 1adsstreated mice developed colitis as evident from thebody weight loss fig 1b increased colon weightlengthratio fig 1c increased dai fig 1d increased stooloccult blood score fig 1e increased intestinal bleedingscore fig 1fincreased diarrhea score fig 1g andincreased colon inflammation score fig 1h in micefig clinical parameters of dsstreated mice administered nicotine or anatabine a mice were orally administered nicotine or anatabine or mgkg for a total of days colitis was induced by oral administration of dss in drinking water ad libitum during days b bodyweight changes in mice during the colitis induction and recovery phases body weight changes were calculated as percentage relative to thestarting day of dss treatment day c colon weightlength ratio are represented as mgcm of colon d dai was calculated according to weightloss colon weightlength ratio and intestinal bleeding e stool occult blood score f intestinal bleeding score g diarrhea score h coloninflammatory status data are shown as mean ± sem p p nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg 0cruiz castro of inflammation page of not subjected to dss treatment nicotine and anatabine administration had no significant effect on these parametershowever mice treated concomitantly with anatabine anddss showed a decrease in colitis severity in particular inmice that received concomitant anatabine and dss treatment anatabine improved body weight recovery at mgkg p fig 1b caused a decrease in global dai relative to that in dsstreated mice at daily dose of p and mgkg p fig 1c and reduced the stooloccult blood score at mgkg p fig 1e interestingly nicotine but not anatabine improved the intestinalbleeding score relative to that in dssadministered mice at mgkg p fig 1f the diarrhea score fig 1gand the colon inflammation score fig 1h were notaffected by nicotine or anatabine no variation in waterconsumption was registered across the different experimental groups online resource anatabine reduces dssinduced gene expression changesin the distal colon on a global levelto complement these pathological findings we analyzed the colon transcriptome dsselicited lesions inmost mouse inbred strains including c57bl6 micehave been shown to be more pronounced in the distalcolon [ ] therefore we focused our study on thatportion of the large intestine principal componentanalysis clearly captured the effect of dss treatmenton the first principal component explainedvariance fig 2a while nicotine treatment did notproduce a pronounced effect on the first principalcomponent in the dsstreatment context anatabinetreatmentin combination with dss produced aresponse more similar to that observed in the watercontrols no dssindicating that anatabine had anameliorating effect on dssinduced gene expressionchanges the results of our differential gene expressionanalysis suggested substantial perturbation of the colontranscriptomefdr fig 2bc application of dss in drinking watercaused significant changes in nearly genes incolon tissues addition of nicotine to dsscontainingdrinking water slightly increased the number of differentially expressed genes in contrast addition of anatabine decreased the number of differentially expressedgenes upon dss treatment by approximately fig 2b and c this alleviating effect of anatabinetreatment on dssinduced gene expression profileswas also apparent in the global gene expression heatmap which showed a global reduction of expressionfold changes upon anatabine treatment fig 2d and eof note in the absence of dss anatabine treatmentdid not result in differential expression of genes andnicotine treatment alone had minor effects fdr pvalue online resource upon dsstreatmentanatabine reduces dssinduced inflammatory geneexpression in the distal colonto gain a more mechanistic understanding of the effectsof nicotine and anatabine treatment we further investigated gene expression changes at the level of functionalgene sets and a ucrelevant causal network model gsaofthe reactome database showed changes acrossmultiple functional categories fig 3b the effects onfunctional categories in dsstreated mice administerednicotine appeared rather similar to those in mice treatedwith dss alone whereas administration of anatabineresulted in a general repression of dssmediated effectswe also evaluated the interaction terms betweennicotineanatabine and dss treatment to more directlyfocus on the modulating effect of anatabine and nicotinetreatment on dss effects fig 3a online resource the following six biological categories showed significant interaction terms upon anatabine and dss treatmentsupporting asignificant suppression of dssinduced effects in thepresence of anatabine immune system extracellularmatrix anization protein localization metabolism hemostasis and signal transduction fig 3bof these we further explored immune system extracellular matrix anization and signal transductionas the most relevant categories in ibdana 20dss fdr allfiginteractiontermsp the hierarchical anization of the reactome database allowed us to investigate the underlying functionalchanges in more detail the immune system categoryincludes adaptive immune system cytokine signaling in immune system and innate immune systemin particular within these immune categories cytokinesignaling egincluding il6 family signaling andtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome signal transduction category signaling byreceptor tyrosine kinasesby rhogtpases signaling by transforming growth factortgfbeta family members mapk family signalingcascades integrin signaling signaling by erythropoietin and signaling by gpcr were found to be significantly impacted online resource online resource within the extracellular matrix anization category almost all subcategories were perturbed includingdegradation of the extracellular matrix elastic fiberformation and extracellular matrix proteoglycansonline resource online resource signalingdegradation ofthe extracellular matrix includesbiological processes such as activation of matrix metalloproteinases mmps and collagen degradationto further follow up on the effects of nicotine andanatabine on inflammatory signaling we evaluatedthe perturbation of the ucrelevant tlril1rtnfr 0cruiz castro of inflammation page of fig transcriptomics results of colon biopsy samples a principal component pc analysis the plot displays all samples in the reduced pc1pc2plane covering of the total data variance it allows us to examine the relationships between the various groups and treatmentsremarkably pc1 captured the pure dss effect black arrow while pc2 captured the pure exposure effects of anatabine and nicotine blue andred arrows respectively b volcano plots for individual gene differential expressions the horizontal axis represents the log2 differential expressionfold changes and the vertical axis represents its statistical significance as log10 fdr c number of differentially expressed genes for theselected pairwise comparisons the bar plot displays the number of genes with positive or negative fold changes with corresponding statisticallysignificant fdr ¤ note that the lower fdr values observed for the ana dss comparisons do not necessarily prefigure a reduction ofbiological effects because the statistics underlying the fdrp values also depend on the gene expression variance within the experimentalgroups d highlevel heatmap of the statistically significant differentially expressed genes for the selected pairwise comparisons in order toprovide a comprehensible display of the large foldchange matrix we first normalized its rows to their maximum absolute values andthen reordered them by hierarchical clustering complete linkage method on the basis of their euclidean distances e scatter plot from thecomparisons between the pure and exposuremodified dss effects by using the differential gene expression results the horizontal axis of thescatter plots represents the fold changes of the pure dss effect water dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure anatabinenicotine dss vs water pairwise comparisons in an idealcase included as a reference [first plot] the bestfitted straight line coincides with the diagonal indicated in green and its slope is equal to the transcriptomics effects of anatabine or nicotine exposure were quantified by the slope of the bestfitted straight lines indicated on each plotnic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgnetwork model by using an established networkenrichment approach we inferred the activationstates of the network nodes on the basis of the observedgene expression changes and calculated the overall network perturbation amplitude for each group comparisonfig 3c dss treatment had a strong activating effect onthis signaling network including activation of several coresignaling nodes such as il1rassociated kinase irak1irak4 and myeloid differentiation primary response myd88 online resource heatmap leadingnodes of note the network perturbation induced bydss treatment was reduced only in the presence ofanatabine with this the results of network analysisfurther supported the ameliorating effect of anatabineon dssmediated inflammation whereas no similareffect was apparent upon nicotine treatment 0cruiz castro of inflammation page of fig biological interpretation of the transcriptomics results a schematic representation of the effects of anatabinenicotine exposure as amodification of the pure dss effect the measured combined anatabinenicotine dss effect captured by the pairwise comparisons anatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors dss treatment and anatabinenicotine exposuretogether with the adjusting twofactor interaction term anatabinenicotinedss anatabinenicotine exposure is synergistic with dss treatmentif the interaction term has the same sign as the pure dss effect eg both are positive as in the schema in contrast the relationship isantagonistic if the interaction term has an opposite sign to the pure dss effect b gsa results for the top reactome pathway categories theheatmap displays the gsa scores normalized rowwise to their maximum absolute values as well as their competitive q1 statistical significancethe fdrs were calculated only among the top reactome pathway categories which are sufficiently distinct in their gene content to assumeindependence of their enrichment results c hierarchical representation of the gsa results for all pathways contained in the top reactomeimmune system category and for the twofactor ana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ ] whereas their statistical significance competitive q1 p values ¤ is indicated by black circles around the nodes theactual labels of the nodes ie the reactome pathway names can be found in online resource the treelike structure of the reactomepathway collection enables topdown investigation within the relevant pathway categories by identifying increasingly specific biologicalprocesses ie involving fewer and fewer genes along the longest paths connecting the statistically significant pathways d npa results for thetlril1rtnfr network model the bar plot displays the npa values for the selected contrasts and their statistical significance is indicated bythe three colored asterisks note that by definition the positive npa values depend on the square of the input genelevel fold changes andtherefore might amplify the differences between the contrasts without preserving the additive relationships among them nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgexpressionvaluesfrom theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six leadingedge genesgenes of the gene set with the highestimmunedifferentialsystem extracellular matrix anization and signaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 signaltransduction and immune system categoriesil6signal transduction and immune system categoriesmmp13 extracellular matrix anization categoryserpine1 signal transduction and extracellular matrixanization categories thbs1 thrombospondin signal transduction and extracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase extracellular matrix anization and immunesystem categories qpcr results showed a clear decreasein dssinduced expression of il33 il6 mmp13 serpine1thbs1 and timp1 expression in the presence of anatabineat mgkg thereby confirming the findings obtainedfrom the microarray data fig 0cruiz castro of inflammation page of fig validation of microarray transcriptomic results using qpcr a boxandwhisker plots for the distribution of the qpcr expression levels δcqof the selected genes the boxes represent the quartiles while the whiskers extend to the most extreme data point which is no more than times the interquartile range from the box the horizontal brackets indicate the statistical significance of the corresponding comparisons mean pvalue smaller than respectively b scatter plots comparing the mouse colon differential expression values obtained bymicroarray horizontal axis and qpcr vertical axis the following similarity metrics were indicated beta is the slope of the best interceptfreelinear fit between microarray and qpcr values r2 is the coefficient of determination measuring the goodnessoffit and pval is the pvalueassociated to the null hypothesis beta nic nicotine mgkg ana anatabine mgkganatabine decreases dssinduced proinflammatorycytokine production and promotes il10 expressionnext we sought to evaluate the impact of nicotine andanatabine on the expression of colonic inflammatorycytokines by mapin line with the previous dataanatabine but not nicotine significantly reduced theabundance of dssassociated inflammatory cytokines including il6 kc tnfα il1α and gcsf whereas itincreased the levels of the antiinflammatory cytokineil10 at a daily dose of mgkg fig interestinglyanatabine also increased the abundance of il21 andshowed a clear tendency towards increasing colonic il 0cruiz castro of inflammation page of fig map results for colon biopsies statistical assessment of the differences observed in the abundance of selected cytokines between thestudy experimental groups and water control fold changes in the treatment groups nicotine and anatabine at and mgkg dss relative towater control are illustrated with colors ranging from blue decrease to red increase statistically significant differences on the basis of raw pvalues no adjustment has been made for multiple testing grey cells highlight missing estimates on the observed differences due to lackof cytokine quantifications nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg1 levels fig taken together the map data confirmthe antiinflammatory effects of anatabine in dssinduced colitisdiscussionour study shows that oral administration of anatabinebut not nicotine reduces the clinical manifestations ofdssinduced colitis in a mouse model in line with thesefindings anatabine demonstrated a global downregulatory effect on dssinduced gene expression changes inthe colon whereas the effects of nicotine were morelimited in particular the results of gene expression profiling further supported the reduction of inflammatorysignaling processes upon anatabine treatment includingsuppression of il6 signaling as shown by gsa findingsand tlr signaling as shown by the results of networkperturbation analysis and gsa map also showed asignificant decrease in the abundance of il6 kc tnfαil1α and gcsf and an increase in the expression of il and the antiinflammatory cytokine il10several studies have reported on the antiinflammatoryeffects of nicotine on the dss mouse model of uc subcutaneous administration of nicotine was shown toameliorate tissue injury in dss colitis and il6 expression in cd4t cells via α7nachrs nicotine wasalso shown to decrease the activation of stat3 throughinduction of mir124 in gutinfiltrated lymphocytes andintestinal epithelial cells strikingly alsharari 0cruiz castro of inflammation page of observed that oral but not subcutaneous injection ofnicotine ameliorated intestinalinflammation and colonic tnfα expression in dsstreated mice in spiteof the reported beneficial effects our results do not support a protective effect of orally administered nicotineon dss colitis of note we found that nicotine significantly reduced dssassociated intestinal bleeding whichwas the only clinical parameter affected by this tobaccoalkaloid the vasoconstrictor effects of nicotine are wellstablished in the gut mucosa nicotine decreasesblood flow and cigarette smoking decreases rectalblood flow to normal levels in patients with uc however how changes in blood flow affect the pathophysiology of uc is still unclear the possible therapeuticuse of nicotine to induce remission in uc patients hasbeen evaluated in five clinical studies [] and twometaanalyses [ ] these studies have demonstrated avariable efficacy of nicotine therapy in induction of remission with several studies showing no effect [ ]moreover a high frequency of adverse events increasedthe withdrawal rate in the nicotine group in some studiesthus limiting the therapeutic benefit of nicotine nucleotidebindingto the best of our knowledge the present study isthe first to assess the impact of anatabine on experimental colitis gene expression analysis of distal colonbiopsy specimens highlighted the antiinflammatoryproperties of anatabine in multiple functional categories including immune responses signal transduction and extracellular matrix anization which inturn encompassed several tlr and cytokine signalingpathways genes contributing to the downregulation oftlr cascades included tlr2 tlr4 and tlr6 and anumber of downstream signaling factors shared byseveral tlrs including myd88 ripk2 irak3 irak4and nod1oligomerizationdomaincontaining protein as well as the nuclearfactors elk1 fos cyclic adenosine monophosphatecamp response elementbinding protein creb1activating transcription factor atf1 and atf2 seeonline resource of the respective gene lists for thecytokine signaling pathways the contributing genes included il6 and il6 receptor α ifnγ il4 cxcl10il22 receptor α2 il2 receptor α tnf receptor superfamily member 1a il17α and il17 receptor α jak1jak2 stat3 and stat4 members of the nfκbsignaling pathway also contributed to the overall reducincluding nfκb p65tion in inflammatory cascadesp105 and p100 subunits iκbα and the nfκb activating protein tab3 in agreement with the results oftranscriptional analysis map findings showed a significant decrease in dssassociated il6 kc tnfα il1αand gcsf protein expression and an increase in il10expression in the presence of anatabine strikinglywhile tlr downstream factors modulated by anatabineare shared by most tlrs the majority of cytokineassociated signaling molecules are specific for eachpathway the seemingly pleiotropic regulatory effects ofanatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different proinflammatory signaling cascadesprevious studies using in vitro and in vivo diseasemodels have demonstrated the antiinflammatory effectsof anatabine [ ] paris showed that this pyridine alkaloid reduced the plasma levels of il1 il6and il17a as well as the expression of il1 infγ andtnfα in the spleen of experimental autoimmune encephalomyelitis mice these authors also showedthat anatabine suppressed stat3 and nfκb phosphorylation in the spleen and brain of these mice anatabine also prevented lps and tnfαinduced nfκb andstat3 phosphorylation in shsy5y and hek cellshuman microglia and human blood mononuclear cells additionally anatabine prevented lpsinduced il1 expression in human whole blood as well as il1il6 and tnfα production in the plasma kidney andspleen in the lps mouse model phosphorylatedstat3 tnfα and il6 were also downregulated in thepresence of anatabine in a transgenic mouse model ofalzheimers disease our results on the effects of anatabine in the dssmouse model of uc are also in line with the findings ofa substantial number of studies demonstrating the protective effects of natural alkaloids in several animalmodels of colitis [ ] intraperitoneal administrationof the minor tobacco alkaloid and nicotinic receptoragonist anabaseine was shown to reduce tissue damagemyeloperoxidase activity and colonic tnfα expressionin a tnbs mouse model of colitis these mice alsoshowed reduced nfκb activation in lamina propriamononuclear cells while mice administered a nicotinicreceptor antagonist presented worse colitis symptomsthan those treated with tnbs alone the algaealkaloid caulerpin reduces dss colitis by suppressingnfκb activation and subsequently inhibiting the colonicproduction of tnfα ifnγ il6 ifnγ and il17 oral administration of berberine also ameliorates dssinduced colitis and downregulates the expression oftnfα ifnγ kc and il17 in colonic macrophages the plantderived alkaloid nmethylcytisine andthe tea alkaloid theophylline mitigate colitis by downregulating tnfα il1 and il6 expression in dss andacetic acid models of colitis respectively [ ] induction ofthe antiinflammatory cytokine il10 in thepresence of natural alkaloids has also been reportedthus indirubin ameliorates dssinduced colitis by suppressing the expression of colonic tnfα ifnγ and il2and upregulating il10 additionally indole alkaloids caulerpin and isatin have been shown to increase 0cruiz castro of inflammation page of the expression of il10 in dss and tnbs models of ibdrespectively [ ] of note our results show anincrease in the abundance of il21 and a tendencytowards increase in colonic il1 levels in the presenceof anatabine although il21 expression is increased inmany chronic inflammatory disorders genetic deficiencyof il21 is associated with ibd and inhibition of il21in the early phases of some inflammatory disorders exacerbates disease development suggesting the dual role ofil21 in the control of immune responses il21also promotes il22 expression in mucosal tcellsthrough a mechanism involving stat3 retinoidrelatedorphan receptor γt and aryl hydrocarbon receptorthereby helping protect immunodeficient mice from dsscolitis interestingly il21 has been recently shownto induce il1 production in dendritic cells through astat3dependent but nfκbindependent mechanismthereby suggesting a link between il21 and il1 mounting evidence suggests that alkaloidsin particular isoquinoline alkaloids present in traditional medicineherbsexertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i | 0 |
"conceptualization data curation investigation project administration supervision validation visualization writing review and editing Goneim I and Ibraheim A performed data curation Kamal NM was involved in literature review writingoriginal draft writing review and editing Alsofiani F and Alawur A performed literature review and writingoriginal draft All authors had read and approved the final manuscriptInformed consent statement Written informed consent in the patients native language was obtained from her fatherConflictofinterest statement The authors declare that they have no conflict of interestCARE Checklist statement The authors have read the CARE Checklist and the manuscript was prepared and revised according to the CARE Laila M Sherief Department of Pediatric Hematology and Oncology Faculty of Medicine Zagazig University Zagazig EgyptLaila M Sherief Amr Ibraheim Department of Pediatrics Faculty of Medicine Zagazig University Zagazig EgyptEsmael Goneim Department of Pediatric Oncology Tanta Cancer Institute Tanta EgyptNaglaa M Kamal Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Cairo EgyptNaglaa M Kamal Fuad A alsofiani Abdulraouf H Alawur Department of Pediatrics Alhada Armed Forces Hospital Taif Saudi ArabiaCorresponding author Naglaa M Kamal MD Full Professor Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Kasralainy Cairo Egypt naglakamalkasralainyeduegAbstractBACKGROUND thalassemia intermedia TI is one of the hemoglobinopathies It constitutes of thalassemia cases yet being associated with a better quality of life than thalassemia major TMCASE SUMMARY We recently reported the first case of acute lymphoblastic leukemia ALL from Egypt in a child with TM and we herein report the first case of ALL from Egypt in a child with TI In this report literature was reviewed for cases of malignancies associated with TI and the possible factors underling the relationship between the two entities We stress that physicians should have a high index of suspicion of malignancies in thalassemia patients if they present with any suggestive symptoms or signsKey words Acute lymphoblastic leukemia Thalassemia intermedia Children Malignancies Iron overload Hydroxyurea Case reportThe Authors Published by Baishideng Publishing Group Inc All rights reservedWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIChecklist Access This is an access that was selected by an inhouse editor and fully peerreviewed by external reviewers It is distributed in accordance with the Creative Commons Attribution NonCommercial CC BYNC license which permits others to distribute remix adapt build upon this work noncommercially and license their derivative works on different terms provided the original work is properly cited and the use is noncommercial See httpcreativecommonslicensesbync40Manuscript source Unsolicited manuscriptReceived January Peerreview started January First decision April Revised May Accepted June in press June Published online August PReviewer Fujioka K Moschovi MA SEditor Dou Y LEditor A EEditor Li JHCore tip Cases have been reported for malignancies in patients of thalassemia major However rare case reports have been reported for malignancies in patients of thalassemiaintermedia as it is a nontransfusion dependent anemia Physicians should have high index of suspicion to diagnose malignancies in patients with thalassemiaintermediaCitation Sherief LM Goneim E Kamal NM Ibraheim A Alsofiani F Alawur A Acute lymphoblastic leukemia in a thalassemia intermedia child A case report World J Clin Pediatr URL wwwwjgnetcom22192808fullv9i11htm dx105409wjcpv9i11INTRODUCTIONThalassemia represents the most common singlegene disorder worldwide The total annual incidence of symptomatic individuals with thalassemia is estimated at in throughout the world of whom nearly have thalassemia intermedia TI which is intermediate in severity between the milder thalassemiaminor and the more severe transfusiondependent thalassemiamajor TM[]We herein report the first case from Egypt with TI who developed acute lymphoblastic leukemia ALLCASE PRESENTATIONChief complaintsThe reported patient is a 15yearold girl with TI who presented at the age of years with pallor decreased growth rate and decreased activity She had severe microcytic hypochromic anemia with hemoglobin Hb of gdLHistory of present illnessPediatric hematologist workup proved the diagnosis of TI Her Hb electrophoresis showed HbA HbF and HbA2 Genetic molecular testing revealed compound heterozygosity for cd27 GT and cd39 CT mutations Hydroxyurea at a dose of mgkg per day was started in addition to folic acidShe was then followed at the pediatric hematology unit at regular intervals to monitor her tolerance to drug therapy with special attention to hematological toxicity There were no significant side effects during seven years of therapy and the patient showed good response with occasional need for blood transfusions She underwent splenectomy during her late teensHistory of past illnessAt the age of years she developed generalized bone aches abdominal pain persistent fever and dyspnea and so she was referred to our hospitalPhysical examinationOn physical examination there was severe pallor tachypnea tachycardia and hepatomegalyLaboratory examinationsInitial complete blood picture showed a Hb level gdL white blood cell count of 109L and platelets count of 109LSerum electrolytes cerebrospinal fluid analysis and kidney and liver function tests were normal expect for mild elevation of total serum bilirubin which was mgdLSerum ferritin was ngdL Serological studies including EpsteinBarr virus cytomegalovirus human immunodeficiency virus hepatitis C virus and hepatitis B virus were negative Lactate dehydrogenase was UL and serum uric acid was mgdLWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIImaging examinationsHer chest Xray was normal Abdominal ultrasonography revealed hepatomegaly with calcular cholecystitis and bilateral diffuse renal enlargement Echocardiography showed mitral valve prolapse with trivial mitral regurgitationMULTIDISCIPLINARY EXPERT CONSULTATIONThe pediatric haematologistoncologist assessment requested bone marrow biopsy which was carried by the hematopathologistBone marrow examination revealed blast cells in a hypercellular marrow with depressed erythropoiesis and granulopoiesies and normal thrombopoiesis Immunophenotyping showed lymphoblasts that are CD10 positive CD19 positive CD34 positive TDT positive HLADR positive CD13 positive and CD33 positiveCytogenetic examination showed a normal karyotype with a DNA index of and negative t1221 t119 BCRABL or 11q23 translocationsmutationsFINAL DIAGNOSISA final diagnosis of Bacutelymphoblasticleukemia ALL with aberrant expression of CD13 and CD33 was achievedTREATMENTInduction chemotherapy of the total XV protocol with prednisone vincristine Lasparaginase doxorubicin cyclophosamide cytarabine 6mercaptopurine and intrathecal chemotherapy was commencedShe received multiple packed red cell transfusions which eventually led to elevation of serum ferritin to ngdL Thus she was started on oral chelation therapy with deferasirox with no complicationsThe patient eventually went into complete remission She then received consolidation chemotherapy of standard risk of the total XV protocol with times of high dose methotrexate HDMTX 6mercaptopurine and intrathecal chemotherapyShe received multiple packed red blood cell transfusions and other supportive measures during the periods of induction and consolidation The transfusions therapy was given according to the guidelines of pediatric oncologists who usually transfuse if Hb level is less than gdL and if associated with pulmonary or cardiac comorbidities or exposed to invasive procedure and hemorrhage and they transfuse with Hb less than gdL The transfusions were not associated with any complications Deferasirox was stopped in consolidation phase during infusion of high dose methotrexateThe main problem observed during the periods of induction and consolidation therapy was increased requirement of blood transfusions as well as repeated infections as during this period the child received intensive chemotherapy which caused bone marrow suppressionOUTCOME AND FOLLOWUPThe child is still in complete remission while being now in the continuation phase for standard risk week fortyDISCUSSIONWe have recently reported the first case from Egypt with thalassemia major TM who developed ALL[] herein we report similarly the first report from Egypt for a patient with TI who developed ALL to highlight that the coexistence of malignancy and beta thalassemia is not rareA thorough look in literature for previously reported cases of malignancies in WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIpatients with TI revealed only one report in from Turkey on a years old boy with TI who developed ALL[] Other previous reports on malignancies associated with TI described nonHodjkinlymphoma[] chronic myeloid leukemia[] Hodjkin lymphoma[] hepatocellular carcinoma[] and thyroid malignancies[] Table To our knowledge our patient is the second worldwide and the first from EgyptAlthough reported cases of malignancies associated with TI are few but it raises the attention of physicians to have high index of suspicion of malignancies in this group of patients when they present with unexplained new symptoms or proposed symptoms and signs of malignancySpecial concern about management plans in these patients as they usually require more frequent blood transfusions as the chemotherapy causes suppression of the bone marrow which adds to the base line chronic hemolysisIn spite that reported cases of malignancies in TI are scarce which makes our trial to find causal relationship between TI and cancer development beyond the scope of our report but we tried to search literature foe possible contributing factors Those factors cant rise to the level of conclusions and definitely need to be proved and validated by larger prospective cohort with large control groups multicenter worldwide studies addressing all possible hypothesesIndeed the most practical logical thinking about that underlying factors for the development of malignancy in TI is being multifactorial[]In a large multicenter study on thalassemia patients from Iran the proportion of patients with cancer was higher in those with TI than those with TM and respectively[] They explained it by the fact that bone marrow in TM patients is suppressed by the regular transfusions while it is very active with high turnover in those with TI[] They suggested that this can lead to a higher rate of DNA repair faults and mutations with subsequent higher rate of hematological malignancies[]Another potential factor is the prolonged use of hydroxyurea Conflicting data are there regarding its carcinogenic potential Hydroxyurea as an antimetabolite interferes with both DNA synthesis and repair mechanisms with later accumulation of mutations and subsequent chromosomal damage Although no studies have yet investigated the relationship between hydroxyurea and the development of cancers in thalassemia but clinically concerns have been raised regarding its potential leukemogenic potential[] Other authors were against this assumption[] The BABYHUG clinical trial which compared hydroxyurea with placebo treated controls refuted this assumption and did not suggest any increased risk of genotoxicity[]Overall there is no evidence to suggest an increased risk of carcinogenesis in patients with thalassemia with hydroxyurea and further studies will need to be designed to establish any potential relationship[]One more probable factor is that patients with TI being having milder disease than those with TM with fewer blood transfusions might lead to delayed diagnosis and even if diagnosed usually there is underestimation of their iron overload problem and sometimes the deceiving relatively mildly elevated ferritin as compared to TM which has been shown to underestimate the true iron burden in TI patient with ultimate fate that these patients accumulate iron but it usually goes unnoticed unchelated and unmonitored Anemia hypoxia and ineffective erythropoiesis suppress the expression of hepcidin by increasing expression of growth differentiation factor and hypoxiainducible transcription factors with the resultant increased intestinal iron absorption and in turn adds to the problem of iron overload[]The longstanding iron overload with its deposition in different body ans with the wellknown association between excess iron and cancer development can be a predisposing factor for all types of malignancies through direct and indirect effects[]Iron can directly damage DNA by nontransferrinbound iron with the consequent inactivation of tumorsuppressor genes such as p53 or their products The indirect effects include the formation of reactive oxygen species ironinduced lipid peroxidation and altered immune system with decreased immune surveillance suppression of tumoricidal action of macrophages and alteration of cytokine activities TI patients usually survive longer than TM patients with enough time for iron overload to develop[]Some authors suggested that improved management protocols of thalassemia patients have led to increased survival with most of them reaching adult age with the consequent occurrence of diseases associated with long life span like malignancies[] This assumption can partially explain other reports in elder patients but it cant work in our patient and the Turkish one who are teenagersMany authors suggested that the occurrence of malignancies in thalassemia patients could be a pure coincidence or a combination of genetic and environmental factors[]WJCPwwwwjgnetcomAugust Volume Issue 0cTable Previously reported cases of thalassemia intermedia who developed malignanciesSherief LM ALL in a child with TINumber of patientsType of malignancyAcute lymphoblastic leukemiaNonHodgkin lymphoma NHLNHL Hodgkin lymphoma HLNHL HL chronic myeloid leukemia CMLCMLHLHepatocellular carcinoma HCCHCCHCCHCCHCCThyroid cancerSome patients have thalassemia major and others have thalassemia intermedia in references and Ref[][][][][][][][][][][][]We can sum up to a clear message that whatever the pathogenesis of malignancies in thalassemias the most important message is to alarm physicians to have high index of suspicion for malignancies if their thalassemia patients develop suggestive symptoms and signs Worsening anemia leukocytosis fever boneache lymphadenopathy and splenomegaly are alarming to look for leukemias and other hematological malignanciesREFERENCES Galanello R Origa R Betathalassemia Orphanet J Rare Dis [PMID ]Sherief LM Kamal NM Abdelrahman HM Hassan BA Zakaria MM First report of acute lymphoblastic leukemia in an Egyptian child with thalassemia major Hemoglobin [PMID ]TuÄcu D KarakaÅ Z G¶k§e M AÄaoÄlu L Un¼var A SarıbeyoÄlu E Ak§ay A DevecioÄlu O Thalassemia Intermedia and Acute Lymphoblastic Leukemia Is it a Coincidental Double Diagnosis Turk J Haematol [PMID 104274tjh20140068]Chehal A Loutfi R Taher A Betathalassemia intermedia and nonHodgkin's lymphoma Hemoglobin [PMID 101081hem120015025]Benetatos L Alymara V Vassou A Bourantas KL Malignancies in betathalassemia patients a singlecenter experience and a concise review of the literature Int J Lab Hematol [PMID 101111j1751553X200700929x]Karimi M Giti R Haghpanah S Azarkeivan A Hoofar H Eslami M Malignancies in patients with betathalassemia major and betathalassemia intermedia a multicenter study in Iran Pediatr Blood Cancer [PMID 101002pbc22144]Alavi S Safari A Sadeghi E Amiri S Hematological malignancies complicating thalassemia syndromes a single center experience Blood Res [PMID 105045br2013482149]Jabr FI Aoun E Yassine H Azar C Taher A Betathalassemia intermedia and Hodgkin lymphoma Am J Hematol [PMID 101002ajh20478]BnaPignatti C Vergine G Lombardo T Cappellini MD Cianciulli P Maggio A Renda D Lai ME Mandas A Forni G Piga A Bisconte MG Hepatocellular carcinoma in the thalassaemia syndromes Br J Haematol [PMID 101046j13652141200304732x]Mancuso A Sciarrino E Renda MC Maggio A A prospective study of hepatocellular carcinoma incidence in thalassemia Hemoglobin [PMID ]Restivo Pantalone G Renda D Valenza F D'Amato F Vitrano A Cassar F Rigano P Di Salvo V Giangreco A Bevacqua E Maggio A Hepatocellular carcinoma in patients with thalassaemia syndromes clinical characteristics and outcome in a long term single centre experience Br J Haematol [PMID 101111j13652141201008180x]Fragatou S Tsourveloudis I Manesis G Incidence of hepatocellular carcinoma in a thalassemia unit Hemoglobin [PMID ] WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TI Maakaron JE Cappellini MD Graziadei G Ayache JB Taher AT Hepatocellular carcinoma in hepatitisnegative patients with thalassemia intermedia a closer look at the role of siderosis Ann Hepatol [PMID ]Poggi M Sorrentino F Pascucci C Monti S Lauri C Bisogni V Toscano V Cianciulli P Malignancies in thalassemia patients first description of two cases of thyroid cancer and review of the literature Hemoglobin [PMID ]Halawi R Cappellini MD Taher A A higher prevalence of hematologic malignancies in patients with thalassemia Background and culprits Am J Hematol [PMID 101002ajh24682]McGann PT Flanagan JM Howard TA Dertinger SD He J Kulharya AS Thompson BW Ware RE BABY HUG Investigators Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia results from the BABYHUG Phase III Clinical Trial Pediatr Blood Cancer [PMID 101002pbc23365] WJCPwwwwjgnetcomAugust Volume Issue 0cPublished by Baishideng Publishing Group Inc Koll Center Parkway Suite Pleasanton CA USA Telephone Email bpgofficewjgnetcom Help Desk wwwf6publishingcomhelpdesk wwwwjgnetcom Baishideng Publishing Group Inc All rights reserved\x0c" | 2 |
Exercise influences the impact ofpolychlorinated biphenyl exposure onimmune functionMahesh R PillaiID1 K Todd Keylock2 Howard C Cromwell3 Lee A Meserve1 Dept of Biological Sciences Bowling Green State University Bowling Green Ohio United States ofAmerica Dept of Exercise Science Bowling Green State University Bowling Green Ohio United States ofAmerica Dept of Psychology and JP Scott Center for Neuroscience Mind and Behavior Bowling GreenState University Bowling Green Ohio United States of America Current address Human Research Protection Program The University of Toledo Toledo Ohio UnitedStates of America lmeservbgsuedua1111111111a1111111111a1111111111a1111111111a1111111111Abstract ACCESSCitation Pillai MR Keylock KT Cromwell HCMeserve LA Exercise influences the impactof polychlorinated biphenyl exposure on immunefunction e0237705 101371journalpone0237705Editor HansJoachim Lehmler University of IowaUNITED STATESReceived April Accepted July Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0237705Copyright Pillai This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data is availableon figshare 106084m9figshare12768332Funding Department of Biological SciencesBowling Green State University Bowling GreenOhio The funders had no role in study design dataPolychlorinated biphenyls PCBs are environmental pollutants and endocrine disruptorsharmfully affecting reproductive endocrine neurological and immunological systems Thisbroad influence has implications for processes such as wound healing which is modulatedby the immunological response of the body Conversely while PCBs can be linked to diminished wound healing outside of PCB pollution systems exercise has been shown to accelerate wound healing However the potential for moderate intensity exercise to modulateor offset the harmful effects of a toxin like PCB are yet unknown A key aim of the presentstudy was to examine how PCB exposure at different doses ppm ipaltered wound healing in exercised versus nonexercised subgroups of mice We examinedPCB effects on immune function in more depth by analyzing the concentrations of cytokinesinterleukin1 IL1 tumor necrosis factorα TNFα Interleukin6 IL6 and granulocytemacrophage colony stimulating factor GMCSF in these wounds inflicted by punch biopsyMice were euthanized at Day or Day after PCB injection n and skin excised fromthe wound area was homogenized and analyzed for cytokine content Results revealed thatwound healing was not signficantly impacted by either PCB exposure or exercise but therewere patterns of delays in healing that depended on PCB dose Changes in cytokines werealso observed and depended on PCB dose and exercise experience For example IL1concentrations in Day mice without PCB administration were less in exercisedmice than mice not exercised However IL1 concentrations in Day mice administered ppm were greater in exercised mice than not exercisedmice Changes in theother measured cytokines varied with mainly depressions at lesser PCB doses and elevations at higher doses Exercise had diverse effects on cytokine levels but increased cytokine levels in the two greater doses Explanations for these diverse effects include the useof young animals with more rapid wound healing rates less affected by toxin exposure aswell as PCBmediated compensatory effects at specific doses which could actually enhanceimmune function Future work should examine these interactions in more detail across adevelopmental time span Understanding how manipulating the effects of exposure toPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functioncollection and analysis decision to publish orpreparation of the manuscriptCompeting interests The authors have declaredthat no competing interests existenvironemntal contaminants using behavioral modification could be very useful in certainhigh risk populations or exposed individualsIntroductionPolychlorinated biphenyls PCB are environmental contaminants that were manufacturedand used in large quantities for over years because of their wide range of applications However eventually the harmful effects of these chemicals were observed and their commercialproduction and use has been prohibited in the US since the 1970s [] Aroclor whichwas administered to mice in the present study is a mixture of PCB congeners with chlorination and was widely manufactured and released into the environment [] Because of theirvurtually indestructible nature PCBs remain in the environment and are known endocrinedisruptors [] PCB exposure has been shown to cause carcinogenecity genotoxicity reproductive toxicity and immunological effects [] The majority of studies indicate that PCBexposure leads to immunosuppression in mice and humans however some studies haveshown an increase in immunological response [] Exposure to polychlorinated biphenylsPCBs has been shown to have immunotoxic effect and to impair the functioning of severalimmune responsive cells [] Previous studies have revealed that PCB can hamper woundhealing [] However these earlier studies were done using an invertebrate model egearthworms with the PCB absorbance through the skin and the wound sizes were measuredonly at hours after wound creation We expanded the present study by examining a wellestablished mouse model and extending the time duration of the wound healing analysisThe present study expanded in another novel way by exploring the impact of exercise onimmune function in animal subjects exposed to PCB Researchers are slowly coming to a consensus that physical activity produces a Ushaped response curve with both no and extremephysical activity having deleterious effects whereas moderate physical activity has beneficialeffects on both overall health and immune system function [] Moderate intensity exercisehas been shown to provide beneficial effects retarding development of various chronic conditions like cardiovascular disease atherosclerosis and chronic inflammation and it also reducing the progression of these diseases and certain neoplasms if they are already present []The production of greater concentrations of antiinflammatory cytokines as a result of exerciseand this production has been demonstrated and this is essential for the positive effects of exercise [] The effect of exercise has also been studied in older mice in which the immune function is suboptimal resulting in greater susceptibility to and delayed recovery from infectionOlder animals also have also been found to have delayed wound healing Moderate intensityexercise in these animals improved wound healing rates It also reduced the concentrations ofproinflammatory cytokines in the wound tissue of these animals []The first aim of the present study was to determine whether PCB exposure impairs woundhealing and if exercise can reduce some of the negative PCB effects and improve wound healing To examine the relationship between PCB exposure and exercise in more detail we measured the concentrations of various cytokines in wound tissue of mice moderately exercised ornot exercised with or without PCB exposure Various immune responsive cells and the cytokines released by them play an important part in the inflammatory phase of wound healingby not only recruiting other immune cells but also in the reepithelization process [] Previous studies have shown that excessive inflammation with the presence of increased proinflammatory cytokines can delay wound healing especially in aged animals [] and thatPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionimmunologically suppressed mice lacking macrophages and neutrophils experience greaterrates of wound healing [] Furthermore it has been shown that exercise improves woundhealing rate in obese and aged mice [] The concentrations of proinflammatory cytokineslike TNFα KC and MCP1 have been observed to decrease in aged mice after exercise andthese findings correlated with more rapid wound healing rates in these animals Howeverthis study did not find significant positive effects of exercise on wound healing in youngermice [] In an evolutionarily less complex species PCB administration in earthworms hasimpaired wound healing and increased allograft rejection [] Additionally in vitro studieshave found PCB to cause enhanced stimulation of neutrophils to produce reactive oxygen species [] However the function of human and mouse macrophages and mouse splenocyteshas been shown to be impaired by PCB administration [] No previous studies havedetermined the amounts of cytokines in wound tissue of animals administered PCB The cytokine data from the present study will give us a novel insight into whether exercise can modulate PCB induced modification of cytokine concentrations and thus negating some of theimmunotoxic effects of PCBMaterials and methodsAnimal careFemale C57BL6 mice weeks of age were obtained from Harlan Laboratories IndianapolisIN and housed in the Bowling Green State University BGSU animal facility All the animalstudies were conducted as approved by the BGSU Institutional Animal Care and Use Committee IACUC under protocol no The mice were individually housed in shoe box cagesand maintained on a reverse lightdark cycle They were provided food Teklad Mouse BreederDiet Envigo Madison WI and water ad libitum and their body weights were recordeddaily After PCB exposure exercise regimen and wound generation the animals were frequently monitored for evidence of distress poor grooming frantic appearance poor coatcondition aversion to handling and wound infection No signs of distress were observed inanimals in this study At the end of testing animals were euthanized by rapid CO2 inhalationfollowed by decapitation with a guillotinePCB exposureAll the animals were given weeks to acclimatize to the animal facility before the study beganAll the animals in the present study both not exercised exercised groups were administeredPCB Arochlor Accustandard Inc New Haven CT USA dissolved in corn oil via intraperitoneal injection at a volume of μlg body weight PCB doses administered were PCB PCB PCB or PCB μgg Previous studies have used similarPCB dosage [] and intraperitoneal injections [] Following acclimatization the animals inthe group not exercised were housed in the animal facility for weeks without exercise andPCB injection was administered days after this 3week period without exercise was overFollowing acclimatization the animals in the exercised group began moderate exercise for a3week period days after completion of the exercise period they were administered PCBExercise regimenThe animals not exercised remained in cages without exercise during the 3week period Thecarts holding the cages of these animals were tethered to the table containing the motorizedtreadmill using large metal clamps so that these no exercise animals not exercised wereexposed to the same sounds and vibrations as the animals in the exercise group The animalsPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionin the exercised group were exercised by running on a motorized treadmill JogADog modelDC6 1HP which was adapted with individual lanes for mice This running was done for minutes daily five days a week for weeks and consisted of moderate intensity running at mmin with a incline These exercise regimens were carried out at the beginning of theactive period of these animals as they are on a reverse lightdark cycle Previousstudies have shown that this intensity of exercise effectively alters immune function []Wound creation and wound size measurementThis procedure was carried out in the animals used in Aim I Table of the study Woundswere made two days following PCB exposure Mice were anesthetized with isoflurane Isoflo1 through a cone mask in oxygen at a flow rate of Lmin during wound creationMice were then administered μgg of analgesic Buprenex Buprenorphine hydrochloride mgml Reckitt Benckiser Healthcare UK Ltd Hull England A similar dose of Buprenex was administered twice daily for days post wounding A Wahl Peanut Hair TrimmerSterling IL was used without guides to remove the hair over an area on the upper back alocation inaccessible to the animal for the creation of wounds The shaved area was thencleaned with Betadine and ethanol Wounds were made using a mm sterile disposablepunch biopsy instrument Robbins Instruments Chatham NJ to create one full thicknessdermal punch resulting in two wounds A Canon EOS Rebel XTi camera was used to photograph the wound at the same time daily until the wounds were reduced to of original sizeor for two weeks after the wounds were made whichever came first Image J software version141o NIH was used to analyze the wound and compare it to the reference wound thus allowing comparison between rates of wound healingEuthanasiaThe animals were euthanized by rapid CO2 asphyxiation followed by decapitation with a guillotine after the wound size reached of the original wound or two weeks post woundingwhichever came first A mm punch biopsy instrument was used to harvest the wound andthe surrounding tissueWound cytokine analysisIL1 IL6 keratinocyte chemoattractant KC monocyte chemoattractant protein1 MCP1and tumor necrosis factor α TNFα protein concentrations in wound tissue Table weredetermined using a BioPlex Pro Mouse Cytokine 23plex Assay kit BioRad LaboratoriesInc Philadelphia PATissue extraction procedureThe wound and the surrounding tissue from animals that were euthanized by CO2 asphyxiation followed by decapitation with a guillotine at Day and Day after wound creation wereTable Detailed distribution of the mice used for wound healing studyWound HealingPCB doses μggTotal101371journalpone0237705t001Number of AnimalsNo ExerciseExercisePLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionTable Detailed distribution of the mice for cytokine analysis studyPCB doses μggTotalDay Day Number of AnimalsNo ExerciseExerciseNo ExerciseExercise101371journalpone0237705t002harvested using a mm punch biopsy instrument Robbins Instruments Chatham NJ Previous study has measured cytokine levels in wound tissue at and days after wound creation[] The tissue was flash frozen in liquid nitrogen and then stored in ËC freezer This tissuewas homogenized using a protocol developed by Frank and Kampfer [] The wound tissuewas homogenized in an extraction solution containing sterile 1X PBS and an antiproteasebuffer cOmplete EDTAfree Protease Inhibitor Cocktail Roche Diagnostics GmbH Mannheim Germany One tablet of cOmplete was added to ml of 1X PBS to prepare the extraction solution A single wound tissue sample was added to ml of the extraction solution andhomogenized using a PowerGen Generator PowerGen Fisher Scientific Pittsburg PAand a saw tooth blades 7X95mm The homogenate was centrifuged at rpm for min atËC The supernatant thus obtained was aspirated into ml syringes BD Biosciences Mexicoand passed through a μm Sartorius Minisart Syringe filter Supelco Bellfonte PA The filtrate approx μl was aliquoted into tubes and stored at ËC A μl aliquot was usedfor protein assayProtein assaysProtein concentrations of tissue extracts were determined by using BioRad Protein AssayDye Reagent Concentrate following the protocol of BioRad Laboratories Richmond CAProtein assays were done in order to express cytokine concentrations in the samples per milligram of protein Stock protein standard solution of 1mgml was prepared by using bovinealbumin purchased from Sigma Chemical Co St Louis MO dissolved in distilled waterBlank and serial dilutions of standard mgml mgml mgml mgml and mgml were used to generate the standard curve The μl aliquot of tissue extract was diluted in μl distilled water total volume μl and μl of this dilution was used to load a single wellof a Costar well plate All protein assays were done in triplicate The dye reagent concentratewas diluted with distilled water and μl of this diluted dye was added to each of thewells The plates were incubated on a shaker for min and then absorbance was measured at nm on a Clariostar plate reader BMG Labtech GmbH Ottenberg Germany and analyzed using Mars Data Analysis Software Version 301R2 BMG Labtech A standardcurve was plotted and used for calculating protein concentrations of tissue extracts The protein concentrations were expressed as mgmlCytokine analysisA BioPlex Pro Mouse Cytokine 23plex Assay kit BioRad Laboratories Inc PhiladelphiaPA was used for measuring cytokine content using a BioPlex instrument lab of Dr Stanislaw Stepkowski The University of Toledo Toledo OH and BioPlex Manager softwareBioRad Laboratories Inc Philadelphia PA was used for machine operation and dataPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functioncollection A BioPlex handheld magnetic washer BioRad Laboratories Inc PhiladelphiaPA was used for all washing steps The protocol accompanying the kit was followed The following cytokines were measured Eotaxin GCSF GMCSF IFNγ Interleukins ILs IL1αIL1 IL2 IL3 IL4 IL5 IL6 IL9 IL10 IL12p40 IL12p70 IL13 IL17A KCkeratinocyte MCP1MCAF monocyte chemotactic and activating factor MIP1α MIP1 macrophage inflammatory protein RANTES regulated on activation normal T cellexpressed and secreted and TNFα tumor necrosis factor alphaData analysisStatistical analysis for all data was completed using SPSS software IBM version Forthe wound size data general linear model Repeated Measures ANOVA along with post hoctests Bonferroni and Tukey was performed for PCB Exercise and Day Wound size on Day day of wound creation over all the groups was normalized to and all the values for thefolowing days were sizes compared to Day Error bars are standard error of the meanSEM Significance was ascribed to p � For the cytokine concentration data general linear model Univariate ANOVA along withpost hoc tests Bonferroni and Tukey was performed for PCB Exercise and Day Based on thesignificance of these data independent Ttests were run between groups within PCB Exerciseand Day Error bars are standard error of the mean SEM Significance was ascribed top � Note Before data were selected for running the statistical analysis in each of thegroups the numbers above or below the mean standard deviation SD were omittedsince there were a few outliers that were drastically skewing the group meansResultsWound healing PCB and exercise effectsDay effects Wounds showed signficant size reductions across days There was a significant main effect of time F6 p0001 There were significant differences for the pairwise comparisons from Day to Day of the analysis see Fig with the wounds decreasingin size in all groupsExercise effects Exercise had no signficant impact on wound healing in the controlgroup PCB However there was a general pattern in which wound sizes decreased at agreater rate in exercised mice administered no PCB Fig 2APCB effects Animals that were not exercised did not experience any significant changesor trends in wound healing rates with varying PCB doses There was a pattern where the ratesof wound healing are greater in PCB administered animals as compared to PCB Fig3C PCB Fig 3E and PCB Fig 3F Comparison of the other PCB doses did not yieldany obvious pattern Fig 3A 3B and 3DExercise and PCB interactions Exercise and PCB exposure appeared to interact indiverse ways In the low dose group exposed to PCB mice Fig 2B those not exercisedhealed at a greater rate than the exercised group A nonsignificant pattern was revealed wherewound sizes decrease at a greater rate in exercised mice administered PCB Fig 2C Additionally wound healing rates appear very similar in exercised and not exercised mice administered PCB Fig 2DNo significant changes or trends in wound healing rates with varying PCB doses wereobserved in animals that were exercised However the figures reveal a pattern where the ratesof wound healing are less in PCB administered animals as compared to PCB Fig 4APCB Fig 4D and PCB Fig 4E No particular pattern was observed with other PCBdoses Fig 4B 4C and 4FPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of the wound size in animals from day day of wound creation to day n Animalsfrom all the treatment groups were combined and used to study the day effect The data have been normalized with theinitial wound size on the day of production Day being As can be expected the wound size decreased consistentlyover the period reduced to less than in days Wound size is significantly less on all the previous days except forthe immediate day before For example wound size on Day is significantly less than Day and Day but not Day All the other days followed a similar pattern101371journalpone0237705g001Fig Effect of exercise on wound size in mice administered varying doses of PCB n Animals that wereadmininstered PCB in varying doses did not show any significant differences or trends in wound size on different daysafter wound production irrespective of whether they were exercised or not However observation of the graph showsthat there seems to be a difference in the mean wound sizes Day through Day between the exercise and no exercisegroups with all doses of PCB except PCB 101371journalpone0237705g002PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of wound sizes on different days in animals not exercised and administered varying doses ofPCB n C E and F The mean wound size is less on Day through Day in PCB treated animals ascompared to PCB PCB and PCB administered mice though no significant differences or trends wereobserved A B and D No significant differences or trends and no difference in means were observed when the otherdoses of PCB were compared101371journalpone0237705g003Cytokine concentrationsThe cytokine concentrations mentioned in the following sections are concentrations in thewound tissue and not circulating concentrations or whole animal concentrationsEffect of exercise and PCB on IL1 levelsIL1 concentrations were determined at Day and Day postwounding Fig UnivariateANOVA revealed following significant interactions PCB X exercise F351 p PCB X day F351 p and exercise X day F151 p Independentttests were performed to further explore the within group interactionsExercise and day interactions The effects of exercise depended on the day that levels ofcytokines were analyzed Concentrations of IL1 on Day in mice not administered PCBwere significantly less in exercised as compared to not exercised mice t5 p PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Comparison of wound sizes on different days in exercised animals administered varying doses of PCBn A D and E The mean wound size is greater on Day through Day in PCB treated animals ascompared to PCB PCB and PCB administered mice though no significant differences or trends wereobserved B C and F No significant differences or trends and no difference in means were observed when the otherdoses of PCB were compared101371journalpone0237705g004Exercised mice not administered PCB revealed significantly less IL1 on Day as comparedto Day �t6 p Fig 5C and 5DPCB and day interactions On Day wound tissue from animals not exercised containedsignificantly less of IL1 in mice given μgg PCB as compared to the animals receiving noPCB t6 p Fig 5A In mice not exercised administered μgg PCB significantly greater concentrations of IL1 were present postwounding on Day as comparedto Day t6 p Fig 5A and 5B On postwounding Day not exercisedmice revealed significantly greater concentrations of IL1 in animals administered μggPCB as compared to mice not administered PCB t4 p Fig 5B Micethat were not exercised on postwounding Day revealed significantly greater concentrationsof IL1 in μgg PCB administered animals as compared to the ones administered μgg PCB t5 p Fig 5B A betweengroup comparison showedPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Effect of exercise and PCB on levels of IL1 in wound tissue at day and day postwoundingComparison of IL1 levels days postwounding in animals not exercised reveals a pattern of reduced concentrationswith different doses of PCB as compared to no PCB administration with PCB demonstrating the most reductionSimilar pattern is observed at Day in exercised animals but with no change in PCB administration However onDay IL1 concentrations in not exercised animals reveals a pattern of dose dependent increase In exercisedanimals on Day concentrations of IL1 are higher in PCB and doses but remain the same with PCB administration101371journalpone0237705g005that postwounding Day mice that were not exercised revealed significantly greater concentrations of IL1 in μgg PCB treated animals as compared to animals administered μgg PCB §t5 p Fig 5B Between group comparison revealed thatmice that were not exercised and administered μgg PCB exhibited significantly greaterconcentrations of IL1 on postwounding Day as compared to Day t6 p Fig 5A and 5BPCB and exercise interactions Tissue from mice administered μgg PCB contained significantly greater concentrations of IL1 on Day in exercised animals as compared to animals not exercised £t6 p Fig 5A and 5C PostwoundingDay animals that were exercised demonstrated significantly lesser concentrations of IL1in μgg PCB administered animals as compared to the ones administered μgg PCB1t7 p Fig 5D On postwounding Day exercised animals demonstrated significantly greater concentrations of IL1 in mice administered μgg PCB ascompared to the animals not administered PCB ¶t6 p Fig 5D On Day mice administered μgg PCB displayed a trend towards less IL1 in exercised as compared to not exercised animals t7 p Fig 5B and 5D Concentrations of IL1 on Day in mice administered μgg PCB were significantly less in exercised mice ascompared to mice not exercised ¥t5 p Fig 5B and 5D Mice that wereexercised on Day postwounding revealed significantly greater concentrations of IL1 in μgg PCB administered animals as compared to the ones not administered PCB Ît6 p Fig 5DPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionFig Effect of exercise and PCB on levels of IL6 in wound tissue at day and day postwounding In notexercised animals on Day there appears to be no difference in the means of IL6 concentrations across PCB dosesHowever Day exercised animals demonstrate a pattern of reduced IL6 concentrations with PCB and PCB but increased with PCB administration as compared to PCB On Day in not exercised mice IL6concentrations appear to be greater with all PCB doses when compared to PCB with PCB being the highestWhereas Day exercised animals reveal pattern of slight decrease with all PCB doses as compared to no PCBadministration101371journalpone0237705g006Effect of exercise and PCB on IL6 levelsIL6 levels were measured at Day and Day postwounding Fig Univariate ANOVArevealed significant PCB effect F356 p It also revealed significant interactionsof the following PCB X exercise F356 p PCB X day F356 p and exercise X day F156 p There was a trend towards PCB X exercise X dayinteraction F356 p Independent ttests were performed to further explorethe within group interactionsExercise and day interactions There were no significant exercise and day interactionsPCB and day interactions On postwounding Day mice not exercised revealed significantly greater concentrations of IL6 in animals administered μgg PCB as compared tomice not administered PCB ¥t7 p Fig 6B Postwounding Day animalsthat were not exercised revealed significantly greater concentrations of IL6 in μgg PCBadministered animals as compared to the ones administered μgg PCB ¶t7 p Fig 6B Mice that were not exercised on postwounding Day demonstratedlower concentrations of IL6 in μGG PCB administered animals as compared to μgg PCB administered mice t7 p Fig 6B In mice that were not exercisedand administered μgg PCB there were significantly greater concentrations of IL6 onpostwounding Day as compared to Day t6 p Fig 6A and 6BPCB and exercise interactions Exercised animals on Day postwounding revealed significantly greater concentrations of IL6 in mice administered μgg PCB as compared tothe animals not administered PCB ¬t7 p Fig 6C Mice that were exercisedPLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionon Day postwounding demonstrated significantly lesser concentrations of IL6 in μggPCB administered animals as compared to the ones not administered PCB t8 p Fig 6C Mice that were exercised on Day postwounding revealed significantlylesser concentrations of IL6 in μgg PCB administered animals as compared to μggPCB administered mice Ît8 p Fig 6C Postwounding Day mice thatwere exercised revealed significantly lesser concentrations of IL6 in μgg PCB treatedanimals as compared to animals administered μgg PCB 1t9 p Fig6C Concentrations of IL6 on Day postwounding in mice administered μgg PCBwere significantly greater in exercised mice as compared to mice not exercised t8 p Fig 6A and 6C In mice that were exercised and administered μgg PCB therewere significantly lesser concentrations of IL6 on Day postwounding as compared to Day �t8 p Fig 6C and 6D In mice administered μgg PCB on postwounding Day revealed significantly lower concentrations of IL6 in exercised animals as comparedto animals not exercised t7 p Fig 6B and 6D On Day postwoundingmice administered μgg PCB revealed significantly lower concentrations of IL6 in exercised animals as compared to animals not exercised £t8 p Fig 6A and 6CEffect of exercise and PCB on KC levelsKC levels were measured at Day and Day postwounding Fig Univariate ANOVA didnot reveal any significant individual or group interactions Independent ttests were performed to explore any within group interactionsFig Effect of exercise and PCB on levels of KC in wound tissue at day and day postwounding KCconcentrations on Day in not exercised animals reveal pattern of reduction with increasing PCB doses except PCB where there is an increase Similar pattern of reduction in KC concentrations with increasing dose is seen on Day exercised animals except for PCB which demonstrates an increase On Day in not exercised animals there is apattern of greater KC with all PCB does as compared to PCB with PCB revealing the highest concentrationsHowever exercised animals on Day demonstrate a pattern of dose dependent reduction in KC concentrations101371journalpone0237705g007PLOS ONE 101371journalpone0237705 August PLOS ONE 0cExercise influences the impact of polychlorinated biphenyl exposure on immune functionExercise and day interactions There were no significant exercise and day interactionsPCB and day interactions Animals not exercised on Day postwounding demonstrated significantly greater concentrations of KC in mice administered μg | 2 |
ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting and Irene von Luettichau Edited byKirsten K NessSt Jude Childrens Research Hospital Department of Sports Medicine and Exercise JustusLiebig University Gieen Gieen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Childrens Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Childrens Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deï¬cits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patients cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modiï¬cations in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reï¬ecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deï¬citsof ï¬ne and gross motor skills reduced muscle strength andpoor physical ï¬tness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe ï¬ndings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months AprilJune at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insuï¬cient knowledgeof the German language and absence of written informedconsent The attending physician conï¬rmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classiï¬ed in underweight 10thpercentile normal weight 10th90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inï¬ated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participantsleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed ï¬exibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand ï¬exibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coeï¬cientÏ were classiï¬ed according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± years¥ years Period postdiagnosis years ± Period posttreatment years ± year years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Signiï¬cant values p ¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classiï¬ed in underweight 10th percentilenormal weight 10th90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deï¬cit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Signiï¬cantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceÏSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ± ± ± ± ± ± ± ± ± All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and ï¬exibility the absolute differences were used as the measured values would have ï¬uctuated around zero and would have givenoversized percentagesÏ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceÏSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ± ± ± ± ± ± ± NMean ± SD Median pvalue ± ± ± ± ± ± ± ± All results were compared to the reference values of each single test item Ï Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and ï¬exibility the absolute differences were used as the measured values would have ï¬uctuated around zero and would have givenoversized percentagesDue to insufï¬cient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ¤ and hand grip strength on the right hand p The performance of eyehand coordination speed ï¬exibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine inï¬uencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly Ï Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma ¦ bone tumor ³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination Ï p and ï¬exibility Ï p were also associatedwith a higher BMI However superior values in upper extremitycoordination Ï p muscular explosive strengthÏ p hand grip strength right Ï p left Ï p and muscle endurance ofthe legs Ï p were associated with increasedBMI The test item speed showed no association with BMIÏ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination Ï p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period Ï p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deï¬cits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma ¦ bone tumor ³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports deï¬ned as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our ï¬ndings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating speciï¬c parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl | 2 |
"protein phosphatase 2a pp2a is a serinethreonine phosphatase that serves as a key regulator of cellularphysiology in the context of apoptosis mitosis and dna damage responses canonically pp2a functions as atumor suppressor gene however recent evidence suggests that inhibiting pp2a activity in tumor cells mayrepresent a viable approach to enhancing tumor sensitivity to chemoradiotherapy as such inhibition can cause cellsto enter a disordered mitotic state that renders them more susceptible to cell death indeed there is evidence thatinhibiting pp2a can slow tumor growth following radiotherapy in a range of cancer types including ovarian cancerliver cancer malignant glioma pancreatic cancer and nasopharyngeal carcinoma in the present review we discusscurrent understanding of the role of pp2a in tumor radiotherapy and the potential mechanisms whereby it mayinfluence this processkeywords protein phosphatase 2a conventional tumor radiotherapy dna damage response radiosensitizationeffectstherapeutic outcomesintroductionwhile mainstays of tumor treatment efforts conventional radiotherapy and chemotherapy often yield unsat[] these poorisfactoryoutcomes are generally linked to tumor cell multidrugresistance and resistance to ionizing radiation [] inaddition while these treatments are welltailored to killing rapidly proliferating tumor cells they generally failto impact hypoxic and quiescent cells ultimately resulting in treatment failure and tumor recurrence [] understanding the mechanistic basiscellchemoresistance and radioresistance is thus vital interestingly recent research evidence suggests that radiosensitization can be achieved by accelerating cell cycleprogression in quiescent cells such that they becomeproliferative [ ] inhibiting proteins such as pp2acan drive quiescent tumor cells to enter mitosis in turnpotentially increasing tumor cell sensitivity to treatment[ ] inhibiting pp2a may therefore represent afortumor correspondence baolinqu301163com xiao lei na ma and lehui du contributed equally to this workthe first medical center of chinese pla general hospital department ofradiation oncology beijing p r chinavaluable new approach to promoting tumor radiosensitization in the present review we discuss current research progress pertaining to the role of pp2a in thecontext of tumor radiotherapythe role of pp2a in radiation therapypp2a is a serinethreonine phosphatase that functionsas a tumor suppressor gene it is a complex composed of a core enzyme and a regulatory subunit thecore enzyme pp2ad is a dimer comprised of a kdcatalytic subunit pp2a c and a kd regulatory subunit pr65 or subunit a pp2a has three subunits including subunit a and two subtypes of subunit c α and with each of these subunits exhibiting distinct structural and catalytic activities there are also multiple subtypes of subunit b that serve to control the specificityand localization of pp2a overall there are four familiesof regulatory b subunits capable of binding to the coreenzyme b pr55 b² b56 or pr61 b pr72 and b[] early research suggested thatpr93pr110pp2a functions as a classic tumor suppressor gene thatis downregulated or nonfunctional in many tumor typesincluding lung skin breast brain ovarian cervical and the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0clei hereditas page of colon cancers [] at a functional level pp2a inhibits a range of tumor signaling pathways preventing il2induced jak3 and stat5 activation which isnormally dysregulated in many malignancies pp2acan also interact with the erk2mek and rasraf signaling pathways through direct and indirect mechanisms soas to control their activation given that constitutive rasrafmekerk signaling is a characteristic of many malignant tumor cells [] these highlights another mechanism whereby pp2a can control oncogenesis pp2a canalso mediate proteasome dephosphorization and therebyimpact cmyc which is often constitutively active in thecontext of tumorigenic transformation [ ]tumor metastasis recurrence and radioresistance allrepresent major roadblocks to the effective treatment ofcancer patients [ ] following pp2a inhibitionmany tumors exhibit slower growth increased apoptoticcell death and greater sensitivity to ionizing radiation ashas been observed in the context of nasopharyngeal carcinoma ovarian cancer pancreatic cancer liver cancerand malignant glioma [] in malignant glioma forexample pp2a inhibition increases the frequency ofcells in the m phase of mitosis inhibiting tumor proliferation while driving increased radiosensitivity similarly pp2a inhibition in nasopharyngeal carcinomahas been linked to significant increases in the frequencyof apoptotic cells and g2m arrest likewise inhibiting pp2a in cancer significantly delayed dma damage repair and thereby facilitated more rapid cell deathfollowing irradiation potential mechanisms whereby pp2a influencesradiotherapy outcomesthe role of pp2a in mitosispp2a is a key regulator of normal mitotic processes greatwall kinase inhibited pp2a by small proteins ensaand arpp19 thereby attenuating pp2aregulated cdk1dephosphorylation and promoting mitosis whereas severemitotic defects occur in the absence of greatwall kinase[ ] pp2a also negatively regulates cdk1 activity viaactivating wee1myt1 and by inhibiting cdc25 inhibiting pp2a can also drive the upregulation of moleculesdownstream of cdk1 thereby promoting mitosis thisgreatwall kinasepp2a signaling pathway is thought to bea primary regulator of normal cdk1 functionality in thecontext of mitosis pp2a can also act on other mitoticmediators such as the mitosisspecific kinases plk1which is a key marker of g2m phase arrest followingpp2a inhibition and which interacts with centrosomesduring mitosis [ ] pp2a is also involved in the negative feedback inhibition of plk1 and aurora b therebyregulating the spindle collection checkpoint in order toensure that microtubules are properly connected to thecentromere inhibiting pp2a causes g2m cell cycle checkpointinactivation and alters dna damage repairradiationinduced dna damage can induce cell cycle arrest and dna damage repair that is mediated by dnadamage checkpoint activation irradiationassociateddna damage can lead to g2m checkpoint activation andconsequent g2m phase arrest enabling dna repair tooccur prior to cellular entry into mitosis cdc2cyclinb is a key regulator of this g2m checkpoint as cdc2cyclin b activation is required in order for cells to proceedfrom the g2 phase into the cleavage phase dnadamage is rapidly followed by the phosphorylation and activation of the atr and atm kinases which in turn activate chk2 and chk1 chk2 and chk1 function in partby suppressing the activation of cdc25 family proteinssuch that cdc2cyclin b activation is inhibited followingdna damage cdc2cyclin b activity is thus reducedresulting in cell cycle arrest following drug orradiationinduced dna damage pp2a dephosphorylationcan inhibit plk1 which phosphorylates and activatescdc25 and cyclins involved in the g2m checkpointthereby facilitating cell cycle progression pp2a may thusprevent cells from dividing by inhibiting plk1 moreover inhibition of pp2a showed that radiationinducedinactivation of atr and chk1 kinase phosphorylation ofcdc2tyr15 and inactivation of g2m phase checkpointswhich attenuated radiationinduced g2m arrest therebyenabling tumor cells to enter into mitosis via reducingdna damage repair efficiency and aggravating cellularmitotic disorders inhibiting pp2a promotes g0 stage tumor cell entry intomitosiscdk2 activity has recently been found to govern the proliferation of quiescent cells following mitosis such thatcells enter the g0 phase when cdk2 activity levels arelow regulating cyclin ecdk2 activity at the end of thecell cycle can promote cellular proliferation inadult anisms pp2a has been found to promote cellular quiescence in studies of drosophila eyes andwings researchers have determined that inhibiting pp2aat the end of the cell cycle can induce additional cell division and thereby impair such quiescence in these drosophila the pp2a subunit b56 family member wdb servesas an important regulator of pp2arelated cellular quiescence when pp2a activity is suppressed cells thatwould normally enter the stationary phase instead exhibit robust cdk2 activity ectopic dominant testinghas further revealed that abnormal cyclin ecdk2 activity can promote additional cell cycle progression in thecontext of pp2a inhibition reduced wdbpp2a activity results in abnormally elevated cyclin e levels enabling quiescent cells to pass through the g0 phase andto thereby enter into mitosis increasing cellular 0clei hereditas page of sensitivity to radiotherapy and chemotherapy given theimportant role of tumor cell quiescence as a driver oftumor radioresistance and recurrence in cancer patients inhibiting pp2a may represent a viable means ofpromoting tumor radiosensitivity by driving cells in theg0 phase of the cell cycle to undergo mitosispp2a as a regulator of apoptosispp2a can control apoptosis by influencing both pi3kakt pathway signaling and the expression and activity ofapoptosisassociated proteins in cells with functional bcl2 for example pp2a has been shown to promote bcl2 dephosphorization and to thereby promoteapoptotic cell death [] in contrast in cells that arehighly metabolically active pp2a can dephosphorylateand thereby activate camkii so as to exert an antiapoptotic effect pp2a also modulates the p53 pathway such that it can activate baxnoxapuma and inhibitbcl2 to drive apoptotic death []in the context of the dna damage response the atmsignaling pathway directly activates and stabilizes pp2a byphosphorylating the ubiquitin ligase mdm2 pp2a in turninhibits akt1 pathway activity and thereby suppressesmdm2 activation thus preventing the mdm2mediateddegradation of p53 in the presence of irreversibledna damage pp2a can also directly dephosphorylatep53 stabilizing this protein an inducing cell cycle arrestand apoptosis inhibiting pp2a may therefore be a viable therapeutic strategy in highly metabolically activetumor cells suppressing pp2a activity in cells exhibitingdna damage can also inhibit bax expression and promote the cell cycle studies of combination radiotherapy and pp2a inhibition have highlighted the consequentinhibition of interactions between p53 and pp2a reducingthe role of the p53 pathway in response to dna damageand promoting cellular proliferation and p53independentapoptotic cell death pp2a as a regulator of the wntcatenin signalingpathwaypp2a is capable of inhibiting wntcatenin signalingpathway activity which normally plays importantroles in governing the migration and proliferation ofcells after wnt ligands interact with specific cellsurface receptors the tcf family transcriptional coactivator catenin undergoes nuclear translocation interactswith tcf and modulates target gene expression thisprocess often becomes constitutively activated duringthe early stages of oncogenesis in tumor cells inwhich the wnt signaling pathway is not active cytoplasmic catenin is generally degraded a complexcomposed of apc dvl axin and 3glycogen synthesis kinase can target catenin for degradation however the pp2ac regulatory subunit has also beenshown to play downstream signaling roles in the contextof the wntcatenin signaling pathway aspirinhas also been found to downregulate wntcateninsignaling pathway activity via inhibiting pp2a positive pp2a feedback signaling has also been suggested toalter the wntcatenin signaling pathway in pancreatic cancer and colorectal cancer cell lines thereby stabilizing the activation of this pathway [ ]intestinal cellscurrent clinical approaches to inhibiting pp2a as anapproach to tumor radiosensitizationto date pharmacological inhibition of pp2a has largelybeen dependent upon the use of natural compoundssuch as okadaic acid and anthraquinone thesecompounds however exhibit varying degrees of toxicityin contrast lb100 is a watersoluble pp2a inhibitor thatis less toxic than these other compounds research suggests that while radiotherapy can enhance pp2a activitylb100 pretreatment prior to radiotherapy can suppresspp2a activation while simultaneously enhancing tumorsensitivity to irradiation lb100 has been leveragedin several clinical trials as a pp2a inhibitor owing to itsefficacy and low toxicity in one study of pancreaticcancerfor example lb100 was found to effectivelyradiosensitize pancreatic cancer cells without adverselyaffecting normal small lb100mediated pp2a inhibition has also been shown to prevent radiationinduced rad51 foci formation and homologous recombination repair thereby causing sustaineddna damage in cells following radiation exposure the presence of undifferentiated stemlike tumor cellscapable of undergoing selfrenewal is thought to be oneof the key mechanisms underlying tumor recurrence andtherapeutic resistance traditionalradiotherapy andchemotherapy efforts are largely unable to impact thesecancer step cells as they grow slowly and are largely quiescent [ ] there is recent experimental evidencethat the receptor corepressor protein complex is a primary determinant of the stemlike properties of cancerstem cells in glioma tumors this receptor corepressor protein complex is composed of the receptorcorepressor protein a deacetylase complex steroidshormone receptors and transcription factors that function to control transcription in the context of glial differentiation cytokineinduced ciliary neurotrophicfactor stimulation of glioma precursor cells has beenshown to inhibit receptor corepressor protein complexactivity via aktpi3kmediated phosphorylation of thereceptor corepressor protein inhibition of pp2ausing lb100 resulted in enhanced ak1 activity therebypreventing receptor corepressor protein complex formation and promoting cellular division rendering quiescent tumor cells more sensitive to irradiation 0clei hereditas page of perspectivesinhibiting pp2a has been conclusively shown to enhancetumor cell radiosensitivity however further research isnecessary in order to facilitate the optimal clinical implementation of these experimental findings for examplewhile many studies have assessed the impact of inhibiting pp2a in tumor cells following radiation exposurefew studies have assessed the effect of such inhibition onnormal tissues which may also undergo potential radiosensitization [] differences in pp2a expressionprofiles between normal and tumor tissues are also essential to ensure that tumor cells can be effectively killedwithout causing undue harm to healthy tissues atpresent there are also few specific inhibitors of pp2aavailable to leverage the potential clinical utility ofcombination pp2a inhibition and radiotherapy treatment it is vital that novel highly specific pp2a inhibitorsbe developed the identification of specific inhibitorsthat preferentially targettumor cells while leavinghealthy cells intact would further advance the clinicalapplications of pp2a inhibition it is also important tonote that many studies of pp2a inhibition have focusedonly on single factors [] whereas tumor resistanceand recurrence are multifactorial in nature at presentthere is a dearth of systematic or comprehensive studiespertaining to the mechanisms whereby pp2a inhibitionbolsters the efficacy of radiation therapyconclusionin summary inhibiting pp2a in combination with radiotherapeutic treatment may represent a viable approachto enhancing patient treatment outcomes and preventingtumor recurrence however further research regardingthe mechanisms underlying such combination efficacy isstill required in addition more specific pharmacologicalinhibitors of pp2a must be developed in order toachieve better clinical outcomesabbreviationspp2a protein phosphatase 2a cdk1 cyclin dependent kinase plk1 pololike kinase cdc2 cyclin dependent kinase atm ataxia telangiectasiamutated atr atm and rad3related pi3k phosphoinositide 3kinasemdm2 murine double minute2 dna deoxyribonucleic acidrna ribonucleic acidacknowledgementsnot applicableauthors contributionsxiao lei and na ma designed the study and made the manuscript yanjieliang did the perspective part yanan han and pei zhang helped participatein the review design baolin qu and lehui du participated in the writing ofpaper and revision of manuscript all authors read and approved the finalmanuscriptfundingno funding was receivedavailability of data and materialsthe datasets are available under reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived july accepted august referencessun d chen j wang y ji h peng r jin l wu w advances inrefunctionalization of erythrocytebased nanomedicine for enhancingcancertargeted drug delivery theranostics verma p mittal p singh a singh ik new entrants into clinical trials fortargeted therapy of breast cancer an insight anti cancer agents medchem httpsdoi1021741871520619666191018172926 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withverthandirad21 to regulate mitosis and an development in drosophilasci rep qin s li j si y he z zhang t wang d liu x guo y zhang l li s li q liu pinto bs orrweaver tl drosophila protein phosphatases 2a b' wdb andy cucurbitacin b induces inhibitory effects via cip2app2aakt pathway inglioblastoma multiforme mol carcinog hein al brandquist nd ouellette cy seshacharyulu p enke ca ouellettemm batra sk yan y pr55alpha regulatory subunit of pp2a inhibits themob1lats cascade and activates yap in pancreatic cancer cellsoncogenesis stafman ll williams ap marayati r aye jm stewart je mroczekmusulmane beierle ea pp2a activation alone and in combination with cisplatindecreases cell growth and tumor formation in human huh6hepatoblastoma cells plos one 201914e0214469lv p wang y ma j wang z li jl hong cs zhuang z zeng yx inhibitionof protein phosphatase 2a with a small molecule lb100 radiosensitizesnasopharyngeal carcinoma xenografts by inducing mitotic catastrophe andblocking dna damage repair oncotarget wrd regulate meiotic centromere localization and function of the meis332shugoshin proc natl acad sci u s a cameron bd traver g roland jt brockman aa dean d johnson l boyd kihrie ra freeman ml bcl2expressing quiescent type b neural stem cells inthe ventricularsubventricular zone are resistant to concurrenttemozolomidexirradiation stem cells xiong y lan j huang k zhang y zheng l wang y ye q pp2acupregulates pi3kakt signaling and induces hepatocyte apoptosis in liverdonor after brain death apoptosis pagano ma tibaldi e molino p frezzato f trimarco v facco m zagotto gribaudo g leanza l peruzzo r szabo i visentin a frasson m semenzatog trentin l brunati am mitochondrial apoptosis is induced by alkoxyphenyl1propanone derivatives through pp2amediated dephosphorylationof bad and foxo3a in cll leukemia 0clei hereditas page of sudoh s kawakami h ohta m nakamura s ciliary neurotrophic factorinducedincrease in betaamyloid precursor protein mrna in rat c6 gliomacells biochem biophys res commun li xf li sy dai cm li jc huang dr wang jy pp2a inhibition by lb100protects retinal pigment epithelium cells from uv radiation via activation ofampk signaling biochem biophys res commun huang cy hung mh shih ct hsieh fs kuo cw tsai mh chang ss hsiaoyj chen lj chao ti chen kf antagonizing set augments the effects ofradiation therapy in hepatocellular carcinoma through reactivation of pp2amediated akt downregulation j pharmacol exp ther ho wsc sizdahkhani s hao s song h seldomridge a tandle a maric dkramp t lu r heiss jd lb100 a novel protein phosphatase 2a pp2ainhibitor sensitizes malignant meningioma cells to the therapeutic effectsof radiation cancer lett miao j shi r li l chen f zhou y tung yc hu w gong cx iqbal k liu fpathological tau from alzheimer's brain induces sitespecifichyperphosphorylation and sds and reducing agentresistant aggregationof tau in vivo front aging neurosci lai d chen m su j liu x rothe k hu k forrest dl eaves cj morin gbjiang x response to comment on pp2a inhibition sensitizes cancer stemcells to abl tyrosine kinase inhibitors in bcrabl human leukemia scitransl med 201911eaav0819 yu cq yin lq tu zt liu dw luo wp the regulatory role of dopaminereceptor d1 on pp2a via sumo1 modification eur rev med pharmacol sciliu l huang z chen j wang j wang s protein phosphatase 2a mediatesjskinduced apoptosis by affecting bcl2 family proteins in humanhepatocellular carcinoma hepg2 cells j cell biochem deng x gao f may ws protein phosphatase 2a inactivates bcl2'santiapoptotic function by dephosphorylation and upregulation of bcl2p53binding blood publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations huang b yang cs wojton j huang nj chen c soderblom ej zhang lkornbluth s metabolic control of ca2calmodulindependent proteinkinase ii camkiimediated caspase2 suppression by the b55betaproteinphosphatase 2a pp2a j biol chem koma yi ito a watabe k kimura sh kitamura y a truncated isoform of thepp2a b56gamma regulatory subunit reduces irradiationinduced mdm2phosphorylation and could contribute to metastatic melanoma cellradioresistance histol histopathol azad a storey a chk1 activity is required for bak multimerization inassociation with puma during mitochondrial apoptosis cellcommunication and signaling ruvolo pp a functional role for the b56 alpha subunit of proteinphosphatase 2a in ceramidemediated regulation of bcl2 phosphorylationstatus and function j biol chem dung td day ch binh tv lin ch hsu hh su cc lin ym tsai fj kuoww chen lm pp2a mediates diosmin p53 activation to block ha22t cellproliferation and tumor growth in xenografted nude mice through pi3kaktmdm2 signaling suppression food chem toxicol li hh cai x shouse gp piluso lg liu x a specific pp2a regulatory subunitb56gamma mediates dna damageinduced dephosphorylation of p53 atthr55 embo j jin z wallace l harper sq yang j pp2ab56 a substrate of caspase3regulates p53dependent and p53independent apoptosis duringdevelopment j biol chem park ds yoon gh lee hs choi sc capsaicin inhibits the wntcateninsignaling pathway by downregulating pp2a biochemical biophysicalresearch communications ashrafizadeh m ahmadi z farkhondeh t samarghandian s resveratroltargeting the wnt signaling pathway a focus on therapeutic activities j cellphysiol oct httpsdoi101002jcp29327 | 0 |
"e commonest cause of cancer death in England and Wales with around 38?000 cases diagnosed each year and ?35?000 deaths. Data from the National Lung Cancer Audit (NLCA) demonstrate significant variation in process and outcome measures across England. In 2009 there was a three-fold difference in survival and active treatment rates which persisted following case mix adjustment (Beckett et al 2012). Furthermore reported lung cancer outcomes in the UK are worse than other comparable European countries (Walters et al 2013) and have improved little in recent years (Khakwani et al 2013). It has been estimated that if survival rates were increased to that of the best in Europe around 1300 lives could be saved each year in the United Kingdom (Abdel-Rahman et al 2009). Variation in health care is not unique to lung cancer and addressing unwarranted variation is challenging (Wise 2010). Although external regulation may have a role in some areas this approach is more difficult to apply to the complex pathways involved in lung cancer diagnosis and treatment. Peer review with supported quality improvement offers a promising alternative but the evidence for its effectiveness is limited. The Washington State's Surgical Care and Outcomes Assessment Program utilised a peer support programme to share the best practice which led to a significant reduction in post-operative complications (Kwon et al 2012). Within the United Kingdom the national COPD resources and outcomes project demonstrated that reciprocal peer-to-peer review led to only limited quantitative differences in the quality of services offered (Roberts et al 2012). A qualitative analysis of this study identified a number of barriers to improvement including difficulties in establishing effective working relationships funding changes and service re-design. In 2003 the Institute for Healthcare Improvement described the collaborative model to achieve a breakthrough improvement (Institute for Healthcare Improvement 2003). Collaboratives allow teams working on the same issue to share good practice and innovation permitting others to take these ideas and implement them in the context of their own anisation resources and case mix. Pronovost et al (2006) successfully employed this collaborative approach together with supported quality improvement to implement five evidence-based interventions on the intensive care unit resulting in the reduction in catheter-related bloodstream infections to zero. These studies offer a persuasive proof of concept but the absence of a control group or of patient-specific outcomes measures limits their implementation in other disease areas such as cancer. The aim of the current study is to determine whether a programme of reciprocal peer-to-peer review visits with supported quality improvement and collaborative working can significantly improve lung cancer process and outcome measures and thus reduce unwarranted variation in outcomes. Materials and methods Study design We conducted a prospective randomised controlled trial. Study population One hundred and sixty-two English NHS trusts were identified from the 2008 NLCA annual report. Centres only providing treatment (not diagnostics) orthopaedic hospitals and ambulance trusts were excluded. Invitations to participate were sent to the remaining 152 trusts. Trusts who agreed to participate and who had 2008 NLCA case ascertainment rates of > 50% expected were paired before randomisation on the basis of contrasting results for four key indicators from the NLCA. The indicators were active treatment rates surgical resection rates median survival and the proportion of patients assessed by a clinical nurse specialist. Each trust was colour coded for each indicator red if below the national average and green if above. By placing each trust with its colour-coded indicators on a map we were able to pair trusts on the basis of a contrasting mixture of red and green indicators and a travel time between centres of around 2?h. On the basis of data from the national COPD resources and outcomes project we determined that we would be able to complete 30 peer review visits during the lifetime of the project thus allowing 30 lung cancer multidisciplinary teams (15 pairs) to be randomised into the intervention arm. Randomisation was performed in a blinded fashion by assigning a random number to each pair of trusts and then allocating pairs numbered 115 to the intervention group. The remaining trusts formed either the control group (if they had agreed to participate) or the non-participant group and had no further contact with the study team but continued to submit data to the NLCA as usual. Intervention The study timeline is shown in . Following introductory workshops the multidisciplinary teams within each pair undertook facilitated reciprocal site visits. The visits consisted of observation of the host team's multidisciplinary team meeting three discussion sessions focusing on the functioning of the mulitdisciplinary team meeting the host team's NLCA data and patient experience questionnaire results. The final session aimed to identify the focus of improvement work to be undertaken by the host team. The quality improvement facilitator introduced a structured template for the quality improvement plans and provided a short introduction to using the model of improvement to guide implementation of the plans. Over the next 12 months the quality improvement facilitator provided support via electronic mail telephone and follow-up visits where required. Teams within the intervention group supported each other via mini-collaboratives in the form of web-based teleconferences and two face-to-face workshops. Outcomes Changes in process and outcome were assessed using data from local quality-improving plans and the following indicators from the NLCA: the proportion of patients discussed at a multidisciplinary team meeting histological confirmation rate active treatment rate surgical resection rate the proportion of patients with small-cell lung cancer receiving chemotherapy and the proportion of patients seen by a lung cancer nurse specialist. Patient experience was assessed in the intervention group using a new lung cancer-specific patient experience questionnaire designed in collaboration with the Roy Castle Lung Cancer Foundation. The questionnaire included 11 questions selected with permission from the previously validated 2004 national cancer patient survey. The questions covered the following domains: communication privacy respect and dignity and three free text questions (see Appendix I). Participating teams were asked to distribute 30 questionnaires to patients recently seen in their services. The clinical nurse specialists distributed the questionnaires to patients who anonymously returned them to the Royal College of Physicians. An independent qualitative ethnographic evaluation of the study was undertaken by the Social Science Applied to Healthcare Improvement Research Group at the University of Leicester. Statistical methods Data were tested for normality using the ShapiroWilk test. Baseline NLCA indicators were taken from the 2009 NLCA report and the intervention control and non-participant groups were compared using a ?2- test. The changes in NLCA indicators from 2009 to 2011 were compared using an independent t-test. Patient experience questionnaire responses for each question were labelled and re-coded to separate them into the worst patient experience category (score 1) vs all other responses (score 0). These scores were then summated to create a domain and a total patient experience score with a possible range of 011 whereby a higher score indicates a worse patient experience. Analyses were performed using the statistical software package SPSS (International Business Machines Corp. Armonk NY USA). Funding and ethics The study was funded by a Closing the Gap' grant from the Health Foundation. The National Research Ethics Service confirmed that the study was service evaluation and quality improvement and did not require ethical review. Results One hundred trusts (66%) replied to the invitation to participate and 91 (61%) agreed to participate in the study. Eighty-one trusts had 2008 NLCA data of sufficient quality to allow pairing. Two trusts provided a joint multidisciplinary team allowing 40 pairs of multidisciplinary teams to be created. One pair agreed to act as a pilot and was excluded from further analysis. Of the remaining 39 pairs 15 pairs (31 trusts) were randomised to the intervention group. The remaining 24 pairs formed the control group. During the study two trusts in the control group amalgamated to form one trust so the total number of trusts in the control group was 47 (Figure 2). Quality improvement plans Two hundred and thirty medical professionals from 31 trusts participated in the review visits. Twenty-nine teams submitted a total of 67 quality improvement plans. The issues identified in the quality improvement plans are shown in Table 1. Eighteen teams collected local data to measure impact. An example of such data is shown in Figure 3. This trust identified small-cell lung cancer chemotherapy as an area for improvement. They introduced a number of changes to their diagnostic and treatment pathways including prioritisation of small-cell pathology reporting faxing of the results to the multidisciplinary team coordinator and lung nurse specialist to allow early booking of oncology appointments. These changes were monitored using a run chart that demonstrated a reduction in the time from multidisciplinary team meeting to chemotherapy treatment and an increase in the proportion of small-cell lung cancer patients receiving chemotherapy from 60% in 2009 to 71% in 2011. National lung cancer audit indicators Baseline (2009) NLCA indicators for the intervention control and non-participant groups were similar (Table 2). The mean change for each NLCA indicator from baseline to 2011 in the intervention and control group is shown in Figure 4. The proportion of patients receiving active anti-cancer treatment in the intervention group increased by 5.2% compared with 1.2% in the controls (mean difference 4.1% 95% CI ?0.1 to 8.2% P=0.055). The remaining NLCA indicators improved similarly both in the intervention and control groups. Patient experience In the intervention group patient experience questionnaires were returned by 438 patients from 30 multidisciplinary teams at baseline (return rate 49%) and 372 patients from 27 trusts following the intervention (return rate 41%). Baseline total scores were low (01.31) indicating high levels of patient satisfaction with the care received although there was a statistically significant (P<0.001) variation in results by the multidisciplinary team (Figure 5). In particular the proportion of patients responding yes to the question did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' varied significantly by 57%100% (P<0.001). The total questionnaire scores did not change significantly during the study (0.220.17 P=0.377) however the variation by the multidisciplinary team reduced (Figure 5). Given that the study aimed to bring the standard of the lower performing trusts to that of the best we performed a post hoc analysis for the five trusts with the worst baseline patient experience scores. This demonstrated that the mean total score improved significantly for these trusts from 0.86 to 0.22 P<0.001. The biggest improvement in this group was seen in the proportion of patients responding yes to the question did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' which increased from 75% to 90% (P=0.05). One multidisciplinary team in this group achieved this improvement by using their baseline questionnaire results as a lever to encourage attendance at an advanced communications skills course. The questionnaire domain-specific scores did not change significantly during the study. Of the individual questions a significant improvement was seen in the rating of the quality of information provided as excellent which rose from 53%59% P<0.05. Qualitative evaluation Participants' experiences were overwhelmingly positive. The reciprocal peer-to-peer visits with supported quality improvement were seen as a strong driver to change. The method of pairing multidisciplinary teams was important. In particular pairing teams with different results not just good' with bad' and allowing teams to visit each other's sites to ensure a two-way sharing of best practice. The independent quality improvement facilitator role was seen as crucial to ensure the visits remained focussed and that the engagement with quality improvement plans was maintained. Finally the involvement of senior managers was crucial to the successful implementation of the quality improvement plans. The detailed findings from the independent evaluation of this project have been reported elsewhere (Aveling et al 2012). Discussion Lung cancer outcomes remain relatively poor and reducing unexplained variation is an attractive proposition to promote improvement. There are a number of ways that clinical teams may share best practice and innovative service delivery models however studies formally evaluating their impact are limited. To our knowledge this is the first study to formally test a national quality improvement strategy which aimed to bring the standard of all lung cancer teams to that of the best. We have demonstrated that reciprocal peer-to-peer review with supported quality improvement is both feasible and effective at stimulating local quality improvement activity but had a relatively modest and somewhat disappointing impact on process and outcome measures as measured by NLCA indicators and a new lung cancer patient experience questionnaire. The facilitated reciprocal visits represented a new and unique opportunity for all members of a lung cancer team to exchange ideas in a supported environment and to formally design then implement quality improvement plans. Nearly two-thirds of lung cancer multidisciplinary teams in England agreed to take part in the study and reassuringly baseline NLCA indicators did not differ significantly between participants and non-participants suggesting that the willingness to participate in quality improvement activity is not related to baseline performance. There were a wide range of areas identified for improvement but nearly half of the teams identified multidisciplinary team meeting effectiveness as a key issue. This is not surprising given that these meetings are pivotal in the lung cancer pathway. Live observation of each multidisciplinary team meeting followed by facilitated feedback proved to be a strong driver to improve on problems such as ensuring weekly presence of all the treatment specialists as well as more simple issues such as room layout. The need to streamline diagnostic and treatment pathways was also identified as a common problem. Recent NICE guidance on the management of lung cancer (National Institute for Health and Care Excellence 2011) recommended a paradigm shift in the diagnostic algorithm from performing multiple diagnostic and staging investigations to performing a single test that will provide both diagnostic and staging information. A number of teams within our study were able to introduce such pathways and demonstrate impressive reductions in diagnostic times and more prompt treatment. This together with more effective multidisciplinary team working may have led to the small increase in the active anti-cancer treatment rates seen within the intervention group. However an alternative explanation for the improvement is regression to the mean given that treatment rates in the intervention group were lower at baseline and overall the lack of significant improvement across the range of NLCA indicators in the intervention group was disappointing. One possible explanation for this is the challenge that some participating teams encountered converting enthusiastic quality improvement plans into tangible improvements for patients over a relatively short time period. The qualitative evaluation confirmed that participants often underestimated the time and energy required to implement and sustain change and highlighted the importance of early engagement with hospital managers to maintain momentum (Aveling et al 2012). Alternatively other national lung cancer initiatives implemented at the time of the study may have driven coexistent improvements in the control group. For example the drive to encourage all lung cancer patients to be referred for clinical nurse specialist support has subsequently been shown to increase the probability that a lung cancer patient receives active treatment. Although even small improvements in lung cancer treatment rates are very welcome it is recognised that undergoing investigation for suspected lung cancer generates high levels of patient anxiety and many patients will remain too unwell to benefit from currently available drugs. The assessment of patient experience is therefore of particular importance in lung cancer. This has proved challenging in detailed national cancer surveys owing to the advance in age poor health and short median survival of lung cancer patients. The response rate to our short questionnaire was relatively high at 4149% compared with the 2011 national survey in which only 7% of lung cancer patients responded (Department of Health 2012) but still represents the views of less than half of lung cancer patients and is a relative limitation in terms of generalisability of the results. It was reassuring to note that at entry to the study patients in the intervention group generally rated their experience as highly satisfactory. This may explain the low number of teams who specifically identified patient experience as an area for quality improvement. In terms of assessing the impact of the reciprocal peer-to-peer review visits and supported quality improvement on patient experience it is likely that this high-baseline satisfaction and the lack of patient experience data for the control group limited our ability to detect a significant change. However our results suggest that those teams with poor scores may be able to use patient experience data to promote significant improvements particularly in areas such as communication skills. Further work is required to develop a lung cancer patient experience measure that is both acceptable to patients and able to detect small but clinically important changes in experience. Although similar in name to the national cancer peer review process there are a number of important differences between the reciprocal peer-to-peer review and supported quality improvement process employed in the current study and national cancer peer review. The latter predominantly performs a quality assurance role ensuring that cancer teams meet a minimum standard via compliance with a number of process measures. Support with quality improvement is not provided and site visits are now rarely performed. The qualitative evaluation of our study highlighted the importance of an independent quality improvement facilitator to the success of the peer review visits and the subsequent implementation of the quality improvement plans. Integration of facilitated reciprocal peer-to-peer review and supported quality improvement into national cancer peer review both for lung cancer and other tumour sites is an attractive proposition and requires further study. However our results suggest that this strategy alone is unlikely to have a major impact on lung cancer treatment rates. This phenomenon is not new in lung cancer for example the introduction and NICE approval of gefitinib treatment for the first-line treatment of lung cancer in 2010 was associated with only a 1% increase in active anti-cancer treatment rates over the following year (Health and Social Care Information Centre 2012). Achieving a stepwise increase in lung cancer treatment rates and survival is likely to require a multi-targeted approach including earlier diagnosis streamlined lung cancer pathways new treatments and a reduction in unexplained variation via supported quality improvement programmes. This project was funded by a Health Foundation Closing the Gap award. (grant number: 7797/5557). Appendix I Improving lung cancer outcomes project: patient experience questionnaireWhat is this survey about? This questionnaire asks about your experience of lung cancer treatment and care at the hospital. It was developed in 2010 and it has been used by Lung Cancer Nurse Specialists in 30 hospital across participating in the Improving Lung Cancer Outcomes Project' led by the Royal College of Physicians and several other anisations. The project aims to improve the quality of services and care for people affected by lung cancer. Why should I complete the survey? We need to know your opinion of the current services and care to help improve these for people affected by lung cancer. Your participation in this survey is voluntary and your answers will be treated in confidence. If you choose not to take part in this survey it will not affect the care you receive from the NHS in any way. Please do not write your name and address anywhere on the questionnaire as this information is not required. No information you give in this questionnaire will be shared in a way that allows you to be identified. How to complete the survey and how long it will take. The questionnaire is short and will take 510?min to complete. Please try to answer every question. Please return your questionnaire even if you have not answered every question. If English is not your first language or if you if you have difficulty understanding the questions then please ask a relative or carer to help you complete the questionnaire. Questions or help? If you have any questions please contact your local lung clinical nurse specialist team. Please select one answer to each question by placing a in the appropriate box. There is space at the end of the survey for you to write any comments. This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. Abdel-Rahman M Stockton D Rachet B Hakulinen T Coleman MP 2009 What if cancer survival in Britain were the same as in Europe: how many deaths are avoidable Br J Cancer 101 (Suppl 2 S115 S124 19956155 Aveling EL Martin G Jiménez GarcÃa S Martin L Herbert G Armstrong N Dixon-Woods M Woolhouse I 2012 Reciprocal peer review for quality improvement: an ethnographic case study of the Improving Lung Cancer Outcomes Project BMJ Qual Saf 21 1034 1041 Beckett P Woolhouse I Stanley R Peake MD 2012 Exploring variations in lung cancer care across the UK-the story so far' for the National Lung Cancer Audit Clin Med 12 14 18 22372213 Department of Health2012National Cancer Patients' Experience Survey Programme 2012/13. England. Health And Social Care Information Centre2012National Lung Cancer Audit Report. Institute for Healthcare Improvement2003The Breakthrough Series: IHI's Collaborative Model for Achieving Breakthrough Improvement. Boston. Khakwani A Rich AL Powell HA Tata LJ Stanley RA Baldwin DR Duffy JP Hubbard RB 2013 Lung cancer survival in England: trends in non-small-cell lung cancer survival over the duration of the National Lung Cancer Audit Br J Cancer 109 (8 2058 2065 24052044 Kwon S Florence M Grigas P Horton M Horvath K Johnson M Jurkovich G Klamp W Peterson K Quigley T Raum W Rogers T Thirlby R Farrokhi E Flum D 2012 Creating a learning healthcare system in surgery: Washington State's Surgical Care and Outcomes Assessment Program (SCOAP) at 5 years Surgery 151 146 152 22129638 National Institute for Health and Care Excellence 2011 The Diagnosis And Treatment Of Lung Cancer (Update Of Nice Clinical Guideline 24) Clinical guidelines CG121 London UK Pronovost P Needham D Berenholtz S Sinopoli D Chu H Cosgrove S Sexton B Hyzy R Welsh R Roth G Bander J Kepros J Goeschel C 2006 An intervention to decrease catheter-related bloodstream infections in the ICU N Engl J Med 355 2725 2732 17192537 Roberts CM Stone RA Buckingham RJ Pursey NA Lowe D Potter JM 2012 A randomized trial of peer review: the UK National Chronic Obstructive Pulmonary Disease Resources and Outcomes Project: three-year evaluation J Eval Clin Pract 18 (3 599 605 21332611 Walters S Maringe C Coleman MP Peake MD Butler J Young N Bergström S Hanna L Jakobsen E Kölbeck K Sundstrøm S Engholm G Gavin A Gjerstorff ML Hatcher J Johannesen TB Linklater KM McGahan CE Steward J Tracey E Turner D Richards MA Rachet B ICBP Module 1 Working Group 2013 Lung cancer survival and stage at diagnosis in Australia Canada Denmark Norway Sweden and the UK: a population-based study 2004-2007 Thorax 68 551 564 23399908 Wise J 2010 Health atlas shows large variations in care in England BMJ 341 c6809 c6809 Study timelines. Figure 2 Consort diagram disposal of eligible trusts including screening randomisation and follow-up. Figure 3 Run chart showing the waiting times from the multidisciplinary team meeting to the first treatment for 10 consecutive small-cell lung cancer patients following the implementation of the quality improvement plan at one trust in the intervention group. Figure 4 Mean change in national lung cancer audit metrics from baseline (2009) to 2011. P=0.055 active treatmentintervention vs controls. Intervention n=31 trusts control n=47 trusts and non-intervention (control and non-participants combined) n=66 trusts. Abbreviations: CNS clinical nurse specialist; MDT multidisciplinary team; SCLC small-cell lung cancer. Figure 5 Total patient questionnaire scores by the multidisciplinary team in the intervention group at baseline (pre) and at the end of the study (post). A low score indicates better experience. Each symbol represents the mean score for each trust in the intervention group. The maximum possible score for the questionnaire is 11. Table 1 Quality improvement plan themes Quality improvement plan theme Number of plans Multidisciplinary team effectiveness 31 Diagnostic pathways 13 Treatment pathways 9 Access to clinical nurse specialists 8 Clinical trial recruitment 4 Patient experience 2 Table 2 Baseline (2009) national lung cancer audit indicators Control ( n =47) Intervention ( n =31) Excluded ( n =67) P -value Mean (%) s.e.m. Mean (%) s.e.m. Mean (%) s.e.m. Control vs intervention vs non-participant control vs intervention Case ascertainment 158.1 38.6 122.0 7.2 107.4 3.6 0.220 0.455 Discussed at the MDT meeting 95.2 0.7 93.7 1.7 90.9 1.9 0.155 0.370 Histological confirmation rate 75.7 1.2 76.4 1.8 78.4 1.6 0.409 0.739 Active treatment 59.5 1.2 55.9 2.2 59.5 1.5 0.305 0.131 Surgery (all cases) 13.4 0.6 13.0 0.8 14.2 0.7 0.469 0.648 SCLC (chemo) 65.1 2.2 66.5 3.9 63.3 2.7 0.746 0.733 Seen by CNS 70.3 3.8 76.6 3.2 58.3 4.2 0.007 0.243 CNS present diagnosis 44.0 3.8 49.4 5.4 38.7 3.8 0.237 0.403 Abbreviations: CNS=clinical nurse specialist; MDT=mulitdisciplinary team; SCLC=small-cell lung cancer. Data are shown as mean and s.e. proportion of patients. BMC Cancer BMC Cancer BMC Cancer 1471-2407 BioMed Central 24386906 3893473 1471-2407-14-3 10.1186/1471-2407-14-3 Study Protocol Study protocol of a randomized controlled trial comparing Mindfulness-Based Stress Reduction with treatment as usual in reducing psychological distress in patients with lung cancer and their partners: the MILON study Schellekens Melanie PJ 1 [email protected] van den Hurk Desiree GM 2 [email protected] Prins Judith B 3 [email protected] Molema Johan 2 [email protected] Donders A Rogier T 4 [email protected] Woertman Willem H 4 [email protected] van der Drift Miep A 2 [email protected] Speckens Anne EM 1 [email protected] 1Department of Psychiatry Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 2Department of Pulmonary Diseases Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 3Department of Medical Psychology Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 4Department of Epidemiology Biostatistics and Health Technology Assessment Radboud University Nijmegen Medical Centre Nijmegen The Netherlands 2014 3 1 2014 14 3 3 28 6 2013 19 12 2013 Copyright © 2014 Schellekens et al.; licensee BioMed Central Ltd. 2014 Schellekens et al.; lic" | 1 |
"Insulin shares a limited physiological concentration range with other endocrine hormones Not onlytoo low but also too high systemic insulin levels are detrimental for body functionsMain body The physiological function and clinical relevance of insulin are usually seen in association with its rolein maintaining glucose homeostasis However insulin is an anabolic hormone which stimulates a large number ofcellular responses Not only too low but also excess insulin concentrations are detrimental to the physiologicalbalance Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening theefficiency of insulin signaling insulin resistance this is not the case for most other hormonal actions of insulinincluding the promotion of protein synthesis de novo lipogenesis and cell proliferation the inhibition of lipolysisof autophagydependent cellular turnover and of nuclear factor E2related factor2 Nrf2dependent antioxidativeand other defense mechanisms Hence there is no general insulin resistance but selective impairment of insulinsignaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthaseeNOS Because of the largely unrestricted insulin signaling hyperinsulinemia increases the risk of obesity type diabetes and cardiovascular disease and decreases health span and life expectancy In epidemiological studieshighdose insulin therapy is associated with an increased risk of cardiovascular disease Randomized controlled trialsof insulin treatment did not observe any effect on disease risk but these trials only studied low insulin doses up to IUday Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes fromMendelian randomization studies comparing individuals with genetically controlled low or high insulin productionConclusions The detrimental actions of prolonged high insulin concentrations seen also in cell culture argue infavor of a lifestyle that limits circadian insulin levels The health risks associated with hyperinsulinemia may haveimplications for treatment regimens used in type diabetesKeywords Hyperinsulinemia Insulin resistance Nrf2 Autophagy eNOS Obesity Type diabetes mellitusInflammation Oxidative stress Cardiovascular morbidity and mortalityBackgroundMost endocrine hormones exhibit a window of optimalphysiological concentrations with compromised function of the anism at levels below or above that rangeFor instance subnormal levels of thyroid hormone define the clinical condition of hypothyroidism above normalrepresent hyperthyroidism which usuallyrequires therapy Addisons disease is characterized bylevels Correspondence kerstinkempfwdgzde2WestGerman Centre of Diabetes and Health Duesseldorf Catholic HospitalGroup Hohensandweg Duesseldorf GermanyFull list of author information is available at the end of the insufficient cortisol production while excess synthesis isseen in Cushing syndromeFor insulin we argue here that not only hypoinsulinemiabut also hyperinsulinemia is detrimental to body functionsHypoinsulinemia causes insulindeficient diabetes and thehormonal actions of insulin are necessary for the life of complex anisms [] On the other hand permanently elevatedlevels of insulin may cause disturbance of normal cellularphysiology and an function We describe the molecularbasis of these undesired insulin actions and consequences ofhyperinsulinemia for healthrelevant endpoints such as obesity or cardiovascular diseases The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cKolb BMC Medicine Page of transformingproteins345trisphosphateMain textInsulin signaling pathwaysBinding of insulin to its cognate cell surfacebound receptor causes a conformational change which initiatesa cascade of signaling events Autophosphorylation bythe insulin receptor tyrosine kinase is accompanied bytyrosine phosphorylation of receptor substrates suchas insulin receptor substrate IRS and Src homology domaincontainingSHCproteins Phosphorylation of IRS allows binding ofphosphatidylinositol3kinase PI3K and synthesis ofphosphatidylinositolPIP3which eventually leads to the phosphorylation and activation of the serinethreoninespecific protein kinaseB AKT Upon activation AKT interacts with severalsubstrates which mediate anabolic effects of insulinthese include glucose uptake glycogen synthesis denovo lipogenesis and protein synthesis [] Additionalpathways triggered by the activated insulin receptorcomprise phosphorylation of SHC followed by activathe Rat sarcoma Rasrapidly acceleratedtion offibrosarcoma Rafmitogenactivated protein kinasesignalregulated kinasekinaseERK pathway Theamitogenactivated kinase promoting cell proliferationand further cellular activities including protein synthesis [] Another pathway triggered by the engaged insulin receptor involves activation of NADPH oxidase and subsequent hydrogen peroxidemediated inhibition of phosphatase and tensin homolog PTENwhich is an important negative regulator of PI3Ksignaling [] Fig terminal kinase ERK isMEKextracellularInsulin secretionInsulin secretion by pancreatic islet cells responds tothe level of circulating nutrients such as glucose aminoacids and free fatty acids Sweeteners may further increase carbohydrateinduced insulin secretion A largenumber of endogenous factors contribute to the regulation of cell activity either stimulatory inhibitory orboth contextdependent These include hormones neurotransmitters and immune mediators [] Insulin isessential for maintaining glucose homeostasis primarilyby facilitating the postmeal uptake of glucose intomuscle and fat cells via translocation of the glucosetransporter [] In the absence of dietary glucose supply and after depletion of glycogen stores glucose in circulation primarily comes from gluconeogenesis in theliver If circulating insulin levels are below the concentrations required for stimulating glucose uptake fromthe blood endogenous stores of fat and protein must beused for energy production For the maintenance of lifein the fasting state circulating insulin levels range between approx and pmoll percentile asdetermined for healthy adult persons in the NationalHealth and Nutrition Examination Survey NHANES[] In response to meals with varying carbohydratecontent insulin levels may rise to the range of approx pmoll []Insulin promotes obesityAlmost years ago insulin injections were one of theoptions of therapy in nondiabetic persons suffering fromundernutrition in the context of various diseases Insulindoses were in the range of those applied in type Fig Metabolic signaling of insulin is anabolic Insulin signaling through the insulin receptor engages several pathways and results in ananabolic state of metabolism The canonical pathway via phosphokinases PI3K and AKTPKB promotes glucose uptake and glycogen and lipidsyntheses whereas lipolysis is inhibited in adipocytes as well as hepatic gluconeogenesis In addition AKT kinases activate mTORC1 whichsupports de novo lipogenesis and protein synthesis The insulin signaling pathway via SHC and the MAP kinases MEK and ERK promotes cellproliferation and protein synthesis Another insulin signaling pathway involves NOX4 and the inhibition of PTEN an inhibitor of the PI3KAKT pathway 0cKolb BMC Medicine Page of diabetes and led to increased appetite and weight gain[] Indeed one major function of insulin as an anabolic hormone is to favor energy storage over usageThis is reflected by the finding that insulin infusion mUkgmin significantly inhibits lipolysis in the skeletalmuscle about and even more effective in adiposetissue about [] Doubling fasting insulin levelssuffices to inhibit lipolysis by approx and to promote lipogenesis for both mean insulin concentrationfor effect EC50 of approx pmoll [] At thisinsulin level gluconeogenesis is still ongoing For halfmaximal inhibition of gluconeogenesis insulin concentrations must rise to approx pmoll in arterial circulation In order to stimulate glucose uptake to halfmaximum insulin levels must rise to even higher levelsapprox ten times the fasting insulin concentrations percentiles for stimulating glucose uptake approx pmoll [] Thus a modest rise doubling offasting insulin levels will already substantially inhibit lipolysis and promote lipogenesis while gluconeogenesis isnot yet inhibited Since such small increases of systemicinsulin concentrations are enough for favoring adipogenesis fasting and diurnal insulin levels are a determinantof obesity risk Indeed several data support the obesitypromoting role of insulin for a detailed review see []Fig These include epidemiological studies which foundhigh fasting insulin levels and concomitant insulin resistance in children and adolescents to be associatedwith higher weight gain in later years [] Studies inadults are less consistent [] Pharmaceutical interventions that lower insulin secretion such as treatment withdiazoxide or octreotide led to significant body weightloss [] This fits with the observation that insulintherapy promotes weight gain [] One probable reasonis that insulin levels in the high normal range are closeto EC50 concentrations for inhibition of lipolysis []In mice modest lowering of circulating insulin concentrations by genetic manipulation ofinsulin genescaused resistance to weight gain despite a highfat diet[] Decreasing insulin gene expression in adult micevia partial gene ablation reversed dietinduced obesity[] In men the Hph1 T polymorphism in the insulingene region was found to be associated with higher fasting insulin levels and a more rapid weight gain in obesepersons[] A Mendelian randomization analysisshowed that persons with genetically determined higherinsulin secretion to oral glucose exhibited a higher bodymass index BMI [] supporting a causal relationshipbetween insulin and obesity riskTaken together moderate to high normal levels of insulin in metabolic healthy persons appear to be a riskfactor for the development of obesitytransientElevated insulin concentrations impair cellularfunctionsinsulin toxicityThere is ample evidence thatincreases ofmetabolic or immune mediator levels are benign physiological responses to biochemical challenges such as therise of systemic glucose or cytokines following mealsHowever chronic elevations of such mediators evenwhen modest in amplitude are usually detrimental tocellular functions [] In the case of glucose the termglucose toxicity was coined to describe this phenomenon[] Prolonged conditions of elevated glucose concentrations cause dysfunction of numerous cell types in thebody including beta cells neurons and the endothelium via several pathways including increased oxidative stress and activation of the sorbitol pathway [] As described below there seems to be a similardetrimental outcome oflongterm elevated insulinconcentrations on cellular functions a correspondingterm would be insulin toxicityFig Insulin promotes obesity Several independent types of observations support the conclusion that insulin promotes adipogenesis andobesity For details see description in the general text 0cKolb BMC Medicine Page of When cells are exposed to continuously elevated insulin levels there is a partial downregulation of insulin signaling The resulting insulin resistance is not primarilydue to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as aresult of receptor dysfunction In response to prolongedhyperinsulinemia there is diminished autophosphorylation of the insulin receptor compared to that observedafter shortterm exposure to insulin and subsequentsteps of the PI3KAKT signaling pathway are affected[ ] Consequently in muscle and fat cells there isless AKTstimulated translocation of GLUT to the cellsurface Fig Thus insulin resistance can be seen as aprotective mechanism for preventing excess activation ofglucose transport from the blood despite chronically elevated insulin levels for maintaining glucose homeostasisin vivo and for mitigating metabolic and oxidative stressdue to excess glucose influx [] Limiting glucoseexportfrom the blood does not necessarily requiredampening of insulin signaling During the early weeksof feeding with a high caloric diet mice show decreasedinsulindependent glucose uptake despite unperturbedinsulinstimulated AKT phosphorylation [ ] Fig An interesting aspect is that the partitioning of insulinreceptor isoforms A and B and of hybrid insulininsulinlike growth factor1 receptors among cell types maycontribute to insulin resistance in some tissues but thepathophysiological relevance is unknown []The phenomenon of insulin toxicity partly arises fromthe fact that there are additional cellular responses to elevated insulin levels which are not toned down duringrole ofinsulin resistance Fig These comprise the upregulation of protein synthesis and the accumulation of ubiquitinated or otherwise modified proteins probably dueto insufficient degradation of these polypeptides [] Amajorinsulin signaling via the canonicalmitogenactivated protein MAP kinase pathway RasMEKERK as well as via activation of NADPH oxidase has been observed [] Even some AKTdependentpathways do not appear to be suppressed by insulin resistance such as de novo lipogenesis in hepatocytes orthe upregulation of mechanistic target of rapamycincomplex mTORC1 [] Enhanced activity ofmTORC1 leads to increased protein synthesis and to deteriorated cell functions largely because of suppressedautophagy []Hence chronic exposure of cells to high ambient insulin concentrations causes an imbalance of cellular responses because of the downregulation of some insulinsignaling pathways insulin resistance but not ofothers The resulting functional state of cells is characterized by an unbalanced anabolic activity of insulin favoring protein synthesis while suppressing autophagyThe latter inhibits autophagic removal and turnover ofproteins and lipids which favors cell senescence [] Inshortterm experiments of exposure to high insulinlevels a protective cellular stress response is observedthe unfolded protein response probably due to the accumulation of derivatized proteins in the absence ofenough disposal In experimentally induced or diabetesassociated chronic insulin resistance and hyperinsulinemiathesuch a protectivestressresponse ofFig Signaling of insulin during insulin resistance During insulin resistance signaling through AKT kinases is partially impaired Not all AKTdependent pathways are affected as well as other signaling pathways indicating that insulin resistance is selective Therefore hyperinsulinemiain the presence of insulin resistance promotes anabolic cell activities via the MEKERK pathway and via mTORC1 Although the PI3KAKT pathwayis impaired during insulin resistance and provides only insufficient translocation of GLUT4 for glucose uptake and deficient activation of eNOSthere appears to be a normal activation of mTORC1 In addition to the anabolic consequences of signaling via the MEKERK pathway depicted inthe figure there is enhanced expression of ET1 and PAI1 not shown as well as inhibition of autophagy and of the nuclear factor Nrf2 whichcompromises cell constituent turnover and cell defense mechanisms to radical stress respectively Hyperinsulinemia downregulates glucoseuptake not only via dampening the PI3KAKT pathway insulin resistance but also via as yet unknown other pathways 0cKolb BMC Medicine Page of endoplasmic reticulum to high insulin levels is diminished or absent []Another activity of insulin is the suppression of transcription of the nuclear factor Nrf2 via induction of heterogeneous ribonucleoproteins F and K [] Nrf2 is thecentral regulator ofthe protective response of cellsagainst oxidative and other types of electrophile stress[] Suppression of Nrf2 expression is expected to impair the antioxidant and cytoprotective defense capacityof cells Insulin signaling required for Nrf2 inhibition occurs via the MAP kinase pathway and thus is not mitigated by insulin resistance [] Fig It therefore canbe assumed that hyperinsulinemia increases the susceptibility of cells against oxidative or other electrophilestress caused by environmental insults Prolonged exposure of cells to high insulin concentrations can thereforebe regarded as toxic Indeed exposure to nmoll insulin has been found to cause DNA damage in a numberof cell types including human lymphocytes [ ] Atthe only concentration tested nmoll insulin impairs oxygen radical defense and sensitizes apoptosispathways in human islets [] In the brain of micehyperinsulinemia impairs electrophysiological functionsof neurons and protein turnover causing a transition toa senescent cell state and an accompanying cognitive decline [] The direct toxic property of insulin deservesfurther studyChronically elevated insulin concentrations impair bodyfunctionsLongevityThe above list of detrimental cellular responses to highambientinsulin concentrations suggests concomitantfunctional impairments at the level of the anism Thisfits with the observed impact of insulin on longevityStudies in nonvertebrate model systems such as thenematode Caenorhabditis elegans or the fruit fly Drosophila melanogaster find that moderate to high insulinactivity shortens lifespan [ ] A consistent findingfrom mouse model studies is that decreased signaling ofanabolic hormones like insulin insulinlike growth factor or growth hormone results in a prolonged lifespan[] Disruption of the insulinreceptor substrate genecaused insulinresistance with defects in insulin signaling[] and led to an extension of lifespan by []A knockout of the insulin receptor in adipose tissue ofmice resulted in an increase of lifespan [] Disruption of the Ins1 gene and one of the two mouse Ins2alleles lowered insulin levels by Ins2 miceversus Ins2 controls in aged female mice without altering circulating insulinlike growth factorIGF1levels These aged experimental mice exhibited lowerfasting glucose improved insulin sensitivity and lifespan extension across[]two different dietsConcomitantly the proteome and transcriptome indicated a profile associated with healthy aging An important aspect is that this study selectively addressed insulinOther interventions for promoting longevity or extending healthspan such as caloric restriction not only lowercircadian insulin levels but several additional hormonesincluding IGF1 are also affected []InsulinIGF1 and hybrid insulinIGF1 receptorsshare signaling via PI3K and AKT The subsequent activation of the protein kinase mTORC1 is a major pathway for supporting somatic growth protein synthesisand fertility while impeding autophagy and lifespanSuppression of mTOR signaling by treatment with rapamycin prolongs life in model anisms and mice []In humans hyperinsulinemia in pre type diabetes isassociated with increased mTORC1 activity which mayhave a negative impact on beta cell survival healthspanand longevity []In the Leiden Longevity Studyfollowup of nonagenarians for years showed a strongassociation of low insulin and glucose levels with healthyaging []Since both IGF1 and insulin employ PI3K and AKTfor signal transduction it is difficult to disentangle thecontribution of insulin versus IGF1 to the modulationof longevity In animal models selective downregulationof circulating insulin levels improved the lifespan ofmice and in elderly persons of the Leiden LongevityStudy only insulin and glucose but not IGF1 consistently met all four predefined criteria of healthy aging[ ] Therefore it may be concluded that low circulating insulin concentrations are not only a marker oflongevity but are causally involved in promoting healthspan or lifespan extensionDetrimental combination of hyperinsulinemia with insulinresistanceInsulin resistance is defined as an attenuated effect of insulin on blood glucose homeostasis primarily by less efficient export of glucose from the blood into skeletalmuscle adipose and liver tissue Permanently elevatedinsulin concentrations in the blood are often consideredas an attempt to overcome insulin resistance Indeed induction of insulin resistance by genetic disruption of insulin signaling as well as by increased growth hormonelevels or an inflammatory milieu causes hyperinsulinemia [] The opposite causality is of more relevanceHyperinsulinemia during insulin infusion in humansleads to systemic insulin resistance [] while in vitrohigh ambient insulin concentrations cause an increase ininsulin resistance in isolated adipocytes [] A summaryanalysis of nine studies in rodents and seven trials inhumans confirmed that the first detectable change in thefasting state after feeding a high caloric diet for severaldays is an increase of basal insulin concentrations but 0cKolb BMC Medicine Page of not of blood glucose concentrations or insulin resistance[] Both increased secretion of insulin by à cells anddecreased insulin clearance in the liver contribute to elevated insulin levels postmeal the latter being of primaryimportance in the case of carbohydraterich food []functionincluding relaxation ofThe combination of hyperinsulinemia and insulin resistance appears to promote hypertension and atherogenesis Fig One important molecule for maintainingvesselthe arterialsmooth muscle layeris nitric oxide NO which isgenerated by endothelial NO synthase eNOS Insulinincreases NO production via posttranslational modification of eNOS via PI3KAKT activity howeverthismechanism is suppressed during insulin resistance [] Decreased local NO production impairs arterialsmooth muscle relaxation and concomitant vasodilatation An important factor in this context is the calciumion homeostasis of vascular smooth muscle cells Underphysiological conditions insulin promotes both calciuminflux into the cytoplasm of smooth muscle cells via several ion channels including Ltype and storeoperatedCa2 channels and counterregulatory NOmediated efflux of Ca2 and K ions which prevents calcium ioninduced myosin lightchain phosphorylation andFig Hyperinsulinemia insulin resistance and cardiovasculardisease High insulin concentrations in the blood may occur due togenetic predisposition overnutrition or highdose insulin treatmentof type diabetes Hyperinsulinemia induces insulin resistance as adefense response to maintain glucose homeostasis Converselyinsulin resistance may be directly induced such as by growthhormone or proinflammatory cytokines Hyperinsulinemia andinsulin resistance enhance the risk of cardiovascular disease byinducing endothelial dysfunction suppression of endothelial nitricoxide synthase eNOS and activation and promotion of calcium ioninflux into smooth muscle cells resulting in increased vascular toneenhanced reabsorption of sodium ions in renal tubules adhesion ofmacrophages to the vessel wall and development of arterial lesionswith increased lipoprotein lipase activity and cardiovascular diseaseconcomitant vascular contractility During insulin resistance NO production is impaired while the supportiveeffect of insulin on calcium ion influx via PI3K deltaand possibly the MEKERK pathway and vasoconstriction is still present Fig [ ]At the same time insulin signals through the mitogenactivated protein MAP kinase pathway to upregulatethe expression of endothelin1 ET1 plasminogen activator inhibitor1 PAI1 adhesion molecules and proinflammatory cytokines [ ] The reninangiotensinsystem is activated in the context of endothelial dysfunction and contributes together with decreased NO production and increased ET1 secretion to vascularstiffening and upregulation of vascular tone [] Inthe absence of hyperinsulinemiainsulin resistance thelower insulin levels exert less potential proatherogenicactivities which are counteracted by insulinstimulatedlocal NO production [ ]Elevated insulin levels also increase the risk of hypertension by enhancing renal reabsorption of sodium ionsby several transport systems in different segments of thenephron Fig Signaling of insulin occurs via insulinreceptor substrate IRS2 and is not suppressed duringinsulin resistance while signaling via IRS1 for counterregulatory mechanisms including local NO production isimpaired [ ] These detrimental actions may be mitigated during chronic hyperinsulinemiainsulin resistance [] However a metaanalysis of prospectiveepidemiological studies showed that the pooled relativerisk of hypertension was when comparing the highest to the lowest category of fasting insulin levels and for comparing highest to lowest selective insulinresistance categories calculated as homeostasis modelassessment of insulin resistance HOMAIR []As a consequence of endothelial dysfunction duringprolonged treatment with insulin arterial lesions rich inlipids are formed [] The progression of early fattystreak lesions to plaques is accompanied by the adhesionand proinflammatory activity of macrophages whicheventually develop into foam cells This process is drivenby endothelial and macrophage lipoprotein lipase activity as demonstrated by the observation of less atherosclerosis in mice with inactivated lipoprotein lipase gene[] Lipoprotein lipase activity in macrophages isenhanced with higher insulin levels in vivo but there isno direct stimulatory effect of insulin on isolated macrophages []The concern that hyperinsulinemia might promote arterial disease in diabetic persons developed in the late1960s due to the steady increase of incidences of atherosclerosis in diabetic persons despite improved glycemiaand decreased risk of ketosis due to insulin therapy []Since then a wealth of data supports the observationthatis ainsulin resistance and hyperinsulinemia 0cKolb BMC Medicine Page of marker of increased risk of cardiovascular disease in thegeneral population and in patients with diabetes [] Although observational studies suggested an approximatelylinear relation between the severity of hyperglycemiaand vascular damage severallarge randomized controlled trials have shown that intense glycemic controlper se does not decrease the risk of macrovascularcardiovascular events [] indeed insulin therapy may evenincrease the risk [ ] However these trials werenot randomized for insulin treatment and treatment ofCVD risk factors was not kept similar between patientsubgroups In the United Kingdom Prospective DiabetesStudy UKPDS hyperinsulinemia and insulin resistancewere not mitigated by insulin treatment and fastingplasma insulin levels even rose [] By contrastinUKPDS and other trials [ ] oral treatmentwith the biguanide metformin reduced the risk of cardiovascular events and in parallel decreased insulin resistance and hyperinsulinemiaIn epidemiological studies of type diabetesit hasbeen consistently observed that the addition of insulin tothe treatment regimen or the intensification of insulintreatment result in a higher rate of cardiovascular events[] Fig Indeed it has been shown that therisk increases with increasing insulin dosage [ ]These epidemiological studies may suffer from residualFig Hazard ratio of insulin medication versus different reference medications Shown are adjusted hazard ratios HR for each study with confidence interval Moderate insulin exposure high insulin exposure moderate insulin dose to units per day §high insulin dose units per day 0cKolb BMC Medicine Page of confounding since it is difficult to account for the possibly more advanced disease stage of patients receivinginsulin A higher rate of hypoglycemic events may be anadditional confounder However covariates consideredin the statistical analyses cover a broad range of potential risk factors from different categories SupplementTable Large randomized controlled trials such asUKPDS [] or the Outcome Reduction With InitialGlargine Intervention ORIGIN Trial [] did not observe an increased incidence of cardiovascular diseasewith insulin therapy but these trials focused on lowdose insulin therapy of up to a median of IUday or IUkgday respectively Similar randomized trials ofhigherdose insulin therapy as typicalfor realworldconditions have not been conducted Recent studies ofrealworld clinical settings report mean daily basal insulin doses of close to IUkg in the Canadian REALITY Study for insulinexperienced patients with type diabetes [] and of IUkg in a physician survey inNew York [] In the European multicentre EUTREAT Study mean baseline insulin doses were between and U per day depending on the type of insulin therapy regimen applied [] It can be concludedthat under realworld conditions the majority of insulinexperienced patients with type diabetes receive higherinsulin doses per day than those tried in UKPDS orORIGINIn the absence of randomized controlled trials aMendelian randomization is an appropriate approach oftesting for a causal relationship in humans Mendelianrandomization studies made use of the finding that somegenotypes are associated with high or low fasting insulinlevels When comparing individuals carrying ¥ allelesthat raise fasting insulin levels with those exhibiting genetically determined low fasting insulin levels an increasedrisk of elevated blood pressure cardiovascular disease andtype diabetes was observed [] In two large recentMendelian randomization studies a genetic profile predicting high insulin levels in the blood after adjustmentfor BMI was also associated with increased systolic bloodpressure and risk of myocardial infarction []ConclusionsAs discussed aboveinsulin signaling engages at leastthree different pathways and modifies a large number ofcellular responses Table Transient elevations of systemic insulin concentrations are physiological responsesto dietary stimuli or other challenges such as environmental toxins [] In case of prolonged upregulationof insulin levels such as in response to overnutritionglucose homeostasis is maintained by mitigating insulinsignaling via PI3KAKT for glucose export from theblood into tissues Consequently insulin resistance hasbeen considered as a defense response in order to avoidTable Key messagescid129 Insulin employs at least three different pathways of signal transductionOne pathway involves phosphorylation steps via IRSPI3KAKT anotheris the MAP kinases RasMEPERK and third leads to the activation ofNOX4cid129 Insulin resistance is selective because it partially mitigates the PI3KAKTpathway for limiting glucose uptake and eNOS activation despitehyperinsulinemia but many other hormonal actions of insulin are notsuppressedcid129 Signaling via mTOR and the MEPERK pathway causes suppression ofautophagy and NRF2 leading to deficient turnover and impaired celldefensecid129 Moderate to high normal insulin levels inhibit lipolysis and promotelipogenesisobesitycid129 Insulin resistance and hyperinsulinemia are interdependent Dietinduced hyperinsulinemia precedes insulin resistancecid129 In epidemiological studies insulin therapy of type diabetes isassociated with a higher risk of cardiovascular events or deathcid129 Randomized trials of insulin therapy and associated risks only studieddosages up to IUdaycid129 Mendelian randomization studies found that genetically determinedhigh insulin levels lead to cardiovascular diseasecid129 Suppression of hyperinsulinemia and concomitant insulin resistanceprovides substantial health benefitshypoglycemia [] However other hormonal actions ofinsulin via the MAP kinase MEKERK pathway and inpart via PI3KAKT are no | 2 |
Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these ï¬ndings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insuï¬cient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social ï¬nancialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreï¬ect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Speciï¬cally procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and speciï¬city[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical eï¬cacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBG level ¥ mmolling mgdl or h blood glucose ¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushings syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coeï¬cients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Students ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe MannWhitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r P There was a significant negative correlationbetween OC or CTX and TG rs P rs P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs P OC rs P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Theseï¬ndings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars P lmgp HTPlmgn XTCð½r P HbA1c brs P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearsons correlation coeï¬cient rs Spearmans correlation coeï¬cient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coeï¬cients Standardized coeï¬cients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These ï¬ndings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen ï¬bres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and conï¬rmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliï¬ozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliï¬ozin dapagliï¬ozin and empagliï¬ozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third theuence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespeciï¬c alkalinephosphatase BAP vitamin D or steroids all of which uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its speciï¬city for bone metabolism is lacking [] BAPis a more bonespeciï¬c marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high speciï¬city in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreï¬ects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These ï¬ndings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DMuencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneï¬cial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the ï¬ndings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conï¬ict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientiï¬c Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang IDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and Diabetes Research and Clinical Practicevol pp [] A V Schwartz D E Sellmeyer K E Ensrud Olderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp [] L Forsen H E Meyer K Midthjell and T H Edna Diabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp [] Y Fan F Wei Y Lang and Y Liu Diabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp [] M Janghorbani R M Van Dam W C Willett and F B HuSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides Vitamin D and glycemic control in diabetes mellitus type Therapeutic Advances in Endocrinologyand Metabolism vol no pp [] H Miyake I Kanazawa and T Sugimoto Association ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalciï¬ed Tissue International vol no pp [] E S Siris R Adler J Bilezikian The clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp [] M B Greenblatt J N Tsai and M N Wein Bone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell Markers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp [] H W S Cabral B F G Andolphi B V C Ferreira Theuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp [] P Iglesias F Arrieta M Pi±era Serum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp [] J M Wishart A O Need M Horowitz H A Morris andB E C Nordin Eï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp [] P Szulc P Garnero F Munoz F Marchand and P D Delmas Crosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp [] K G M M Alberti P Z Zimmet and WHO ConsultationDeï¬nition diagnosis and classiï¬cation of diabetes mellitusand its complications Part diagnosis and classiï¬cation ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp [] J StarupLinde and P Vestergaard Biochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp [] L Achemlal S Tellal F Rkiouak Bone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin Association of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira Metabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco Bone turnover markers in men andwomen with impaired fasting glucose and diabetes Calciï¬edTissue International vol no pp [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann Lower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu Signiï¬cant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp [] A Tan Y Gao X Yang Low serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang The relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp [] Y Chen Q Zhao G Du and Y Xu Association betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a | 2 |
" accurate detection of patients with minimal residual disease mrd after surgery for stage ii coloncancer cc remains an urgent unmet clinical need to improve selection of patients who might benefit formadjuvant chemotherapy act presence of circulating tumor dna ctdna is indicative for mrd and has highpredictive value for recurrent disease the medocccreate trial investigates how many stage ii cc patients withdetectable ctdna after surgery will accept act and whether act reduces the risk of recurrence in these patientsmethodsdesign medocccreate follows the trial within cohorts twics design patients with colorectal cancercrc are included in the prospective dutch colorectal cancer cohort plcrc and give informed consent forcollection of clinical data tissue and blood samples and consent for future randomization medocccreate is asubcohort within plcrc consisting of stage ii cc patients without indication for act according to currentguidelines who are randomized into an experimental and a control armin the experimental arm postsurgery blood samples and tissue are analyzed for tissueinformed detection ofplasma ctdna using the pgdx elio¢ platform patients with detectable ctdna will be offered act consisting of cycles of capecitabine plus oxaliplatin while patients without detectable ctdna and patients in the control groupwill standard followup according to guidelinethe primary endpoint is the proportion of patients receiving act when ctdna is detectable after resection themain secondary outcome is 2year recurrence rate rr but also includes 5year rr disease free survival overallsurvival time to recurrence quality of life and costeffectiveness data will be analyzed by intention to treatcontinued on next page correspondence mkoopman6umcutrechtnlsj schraa and kl van rooijen are shared first authorrja fijneman gr vink and m koopman are shared last author1department of medical oncology university medical center utrechtutrecht university heidelberglaan cx utrecht the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cschraa bmc cancer page of continued from previous pagediscussion the medocccreate trial will provide insight into the willingness of stage ii cc patients to be treatedwith act guided by ctdna biomarker testing and whether act will prevent recurrences in a highrisk populationuse of the twics design provides the opportunity to randomize patients before ctdna measurement avoidingethical dilemmas of ctdna status disclosure in the control grouptrial registration netherlands trial register nl6281ntr6455 registered may wwwtrialregisternltrial6281keywords colon cancer circulating tumor dna ctdna adjuvant chemotherapy twics in patients with stage ii colon cancer cc the recurrence rate rr after surgery is approximately disease management after surgical resection in stageii cc is still under debate because the overall survivalos benefit of adjuvant chemotherapy act in thisgroup of patients varies between and only [ ]moreover offering act in a lowrisk population induces an important amount of overtreatment with unnecessary but sometimes severe toxicity and costsseveral prognostic characteristics of stage ii cc havebeen identified to provide better selection of patientsthat might benefit from act patients with presence ofat least one of the following characteristics are classifiedas being at high risk of disease recurrence poorly differentiated histology pt4 lesions inadequately less than sampled lymph nodes lymphovascular or perineuralinvasion or tumor presentation with perforation or obstruction in contrast patients with a deficient mismatch repair dmmr status in stage ii cc have a low risk ofrecurrence and act is not considered beneficial irrespective of the presence of other risk factors [ ]other known prognostic factors in cc like gene expression profiles or braf v600e and ras mutations have been investigated but do not adequatelyidentify the patients that will benefit from act []despite the definition of high and low risk subgroupsof stage ii cc patients retrospective analyses demonstrated that improved survival after administration ofact was not observed in high risk patients or exclusivelyin patients with a pt4 tumor [] therefore in thenetherlands act is currently only recommended in stageii cc patients with a pt4 tumor without dmmrunfortunately also pt4 is not an absolute predictorfor disease recurrence in stage ii patients in a retrospective analysis of stage ii cc patients with pt4tumors the 3year diseasespecific survival rate aftersurgery was in patients who received act and in patients who did not receive act whichmeans that of these patients are exposed to actunnecessarily considering nonpt4 stage ii patients a population registry analysis of patientsshowed that in this group of patients sufferedfrom recurrences these data demonstrate thatusing pt4 as a prognostic factor results in significantunder and overtreatmentminimal residual disease mrd is defined as the presence of tumor cells in the blood bone marrow or lymphnodes not detected by conventional staging procedures patients who have mrd after surgery are not completely cured and therefore at high risk of developingdisease recurrence development of a highly specific andsensitive biomarker testindicative for mrd wouldallow identification of the subset of patients likely to experience recurrence of disease thereby improving the selection of patients who may benefitfrom adjuvanttreatment in adjuvant trials this would solve problemsof high numbers needed for inclusion and dilution of effectiveness of adjuvant treatment by inclusion of manyalready cured participants cellfree circulating tumor dna ctdna has a strongpotential for being this sensitive and yet specific biomarker ctdna consists of smallfragments usually bp of tumorderived dna containing tumorspecific mutations which can be detected in liquid biopsies such as blood samples [] because of the shorthalflife of ctdna estimates ranging from to min the presence of ctdna in blood samples taken several days after surgery presumably reflects a state ofmrd [] patients with mrd have the highest riskfor disease recurrencerecently the presence of ctdna after tumor resection demonstrated a very strong prognostic value fordisease recurrence in stage ii cc with a 2years rrof versus in patients with and without detectable ctdna after surgery respectively in thisstudy the univariate prognostic value of ctdna was muchhigher than that of pt4 status hazard ratio of versus respectively there are several ongoing trials that usectdna in prognostication nct03637686 nct03737539nct03416478 nct03312374 nct02842203 nct0361 and treatment nct03748680 actrn12615000381 nrggi of nonmetastatic cc but to date thereare no results available of randomized controlled trialsrcts that use ctdna for selection of act treatment 0cschraa bmc cancer page of the accumulating evidence for the strong prognosticvalue of ctdna raises an important ethical dilemma forrandomization of patients when designing a conventional rct in which patients with detectable ctdna arerandomized into act treatment or standard of carefollowup while disclosing ctdna status to the controlgroup indeed the knowledge of having a very highchance of disease recurrence will be a big burden for patients with detectable ctdna in the control group andtheir caregivers as they are not being offered any additional therapy this warrants an innovative trial designlike the trialdifferent from the conventional rctwithin cohorts twics design [] the twics design enables an experimental group in which ctdna status is disclosed and a control group that is unaware oftheir ctdna statusthe medocccreate trial is designed as a multicenter twics study with two parallel groups in whichwe will investigate whether stage ii cc patients with detectable ctdna after resection are willing to receiveact and whether act reduces the rr in these ctdnapositive patientsmethodsdesignaimthis study investigates the willingness of patients to receive act after detection of ctdna postsurgery andthe effect of ctdnaguided act on the rr in stage iicc patientsstudy designthe medocccreate trial follows the twics designand is performed within the prospective dutch colorectal cancer cohort plcrc wwwplcrcnl plcrc is set up by the dutch colorectal cancer groupdccg and collects clinical data and patient reportedoutcome measures proms at baseline and at multipletime points during followup fig at enrollment patients give informed consent for use of their clinical dataand optionally for receiving quality of life questionnairesfig schematic presentation of medocccreate using the trial within cohort twics design a plcrc is a nationwide cohort study in thenetherlands with inclusion of crc patients all stages by optional informed consent regarding collection of biomaterials and futurerandomization observational as well as interventional trials can be performed within the cohort b nonmetastatic crc patients are included inmedocc when the patient signs informed consent for plcrc including additional blood sampling blood samples are withdrawn beforeresection days after resection and every months during the first years of followup c eligible stage ii colon cancer patients arerandomized following the twics design in the experimental group informed consent is being asked for immediate ctdna analysis of theblood sample obtained after resection if ctdna is detectable patients are offered adjuvant chemotherapy the control group is not informedabout medocccreate and will receive standard of care 0cschraa bmc cancer page of collection of biomaterials for research additional sequential blood sampling and for being approached forfuture studies conducted within the infrastructure ofthe cohort either in accordance with the twics design or notpatients with pt4 tumors will be offered act therefore we will include eligible patients with pt4 tumors without a recommendation for act according totheir treating physician and use pt4 status as a stratification factorpatient selection and recruitmentpatients will be recruited in both academic and nonacademic hospitals in the netherlands that are participating in plcrc nonmetastatic colorectal cancercrc patients that give informed consent for plcrcincluding consent for additional blood sampling at enrollment will be included in the observational plcrcsubstudy medocc molecular early detection of coloncancer before surgery the participants are eligible forthe current medocccreate trial if they meet the following criteria after surgery histopathological confirmed and radically resected stage ii cc age ¥ years informed consent for plcrc and medoccincluding consent for randomization in future trials anduse of tissue physical condition allows treatmentwith combination chemotherapy consisting of a fluoropyrimidine and oxaliplatin and no indication foract according to the treating physician andor multidisciplinary board patients who are pregnant have hadanother malignancy in the previous years except forcarcinoma in situ or patients with contraindications forfluoropyrimidines andor oxaliplatin will be excludedcurrently the dutch guidelines recommend act forpatients with pt4 tumors however there is large ageand hospital dependent variation in administration ofact in this group and in clinical practice not all stage iiblood sample collectionblood samples are collected before and days aftersurgery for all patients included in the medocc clinicalstudy predominantly comprising stage i ii and iii ccpatients table blood samples two tubes of mlper timepoint are collected in cell free dna streckblood collection tubes for various research purposesamong which the medocccreate trialrandomizationabout week after surgery when the histopathologicalreport is finished medocc patients who are eligiblefor medocccreate will be randomized to theintervention or control arm using slim an onlineplatform to manage patientinclusion including arandomizationgeneratedcomputerservice therandomization schedule is stratified by tstage and usespermuted blocks of random sizes allocation concealment will be ensured as the service will not release therandomization code only patients randomized to theintervention arm will be informed about medocccreate according to the twics design experimental armafter randomization only patients randomized to theexperimental arm will be asked separate informedtable standard protocol items for intervention trials spirit schedule of enrollment interventions and repeated measurementsact adjuvant chemotherapy ctdna circulating tumor dna qol quality of life intervention group only intervention grouponly if ctdna is positive 0cschraa bmc cancer page of consent for the immediate analysis of ctdna status ofthe postsurgery sample a small proportion of patientsestimated approximately will have detectablectdna in their blood these patients will be offeredact patients decide whether they accept or refuse thistreatment patients without detectable ctdna will receive routine standard of careact will consist of months of capecitabine and oxaliplatin capox or months of fluorouracil leucovorinand oxaliplatin folfox treatment starts preferablywithin weeks and not beyond weeks after surgeryduring and after completing act routine followupwill consist of regular visits at the surgical outpatient department blood withdrawals for analysis of carcinoembryonic antigen cea and imaging standard ultrasoundofthe liver according to current guidelines in thenetherlands no additional imaging will be performed toprevent detection biascontrol armin the control arm patients will not be informed aboutthe medocccreate trial and receive routine followup care consisting of cea tests every months for thefirst years and abdominal ultrasound or ct every months in the first year and once a year afterwards oneyear after surgery a colonoscopy is performed postsurgery blood samples will not be tested for ctdna immediately but will be analyzed batchwise after severalmonths without result disclosure to patients and theirtreating physiciansfollowupblood samples will be collected at 6monthly intervalsfor the first years after surgery for both patients in theexperimental arm and the control arm conform themedocc study protocol these samples will not be analyzed for ctdna immediately and results will not bedisclosed to patients and treating physicianstumor tissueinformed ctdna analysisafter surgery the local pathologist will send a formalinfixed paraffinembedded ffpetissue block to thecentral laboratory where dna will be isolated for further analysisthe postsurgery blood sample is drawn between and days after surgery the sample is not withdrawnbefore day to reduce the risk of falsenegative ctdnatests due to the relatively large amount of cell free dnacfdna released due to cell damage after surgery theblood is taken no later than days after surgery to beable to start chemotherapy within weeks after surgerysamples are kept at room temperature and sent by regular mail to the central laboratory within days wherectdna will be isolated for further analysistumor tissue dna will be analyzed by targeted nextgeneration sequencing of a panel of more than genes using the pgdx elio¢ tissue complete assay frompersonal genome diagnostics pgdx baltimore mdusa plasma ctdna will be analyzed by targeted nextgeneration sequencing of a panel of more than genesusing the pgdx elio¢ plasma resolve assay from pgdxbaltimore md usa both panels include the mostcommonly mutated genes in cc including apc tp53kras and braf tumor tissue dna mutations are usedas input information for plasma ctdna mutation callingthereby increasing both sensitivity and specificity of thectdna testprimary endpointthe primary endpoint is the proportion of patients starting with act after detection of ctdna in the postsurgery samplesecondary endpointsthe most important secondary endpoint is 2year rr inpatients with detectable ctdna in their blood expressedas the proportion of patients that experience a recurrence within years after surgery detection of recurrences in months after surgery will occur by standardfollowup investigations including monthly bloodsampling of tumor marker cea and monthly imagingwith ultrasound liver or ct abdomen and when indicated by symptoms radiological andor histopathological evidence is used to confirm the recurrence thedate of the said investigation is considered the date ofrecurrencedata about followup recurrences and survival areroutinely collected within plcrc using the netherlandscancer registry ncr managed by the netherlandscomprehensive cancer anisation iknl to provideinsight in the characteristics and magnitude of cancer inthe netherlands other secondary endpoints include 2year rr in aperprotocol analysis 5year rr intentiontotreatand perprotocol analysis time to recurrence ttr and 5year disease free survival dfs rate and7year diseaserelated os rate and 5year rr inpatients with undetectable ctdna after surgery quality of life qol and costeffectiveness of the ctdnaguided strategytimetoevent outcomesos rate is expressed as proportion of patients that arealive and years after surgery dfs rate is expressed asproportion of patients that did not experience disease recurrence a second primary cc or death within and years after surgery ttr is expressed as time monthsbetween surgery and detection of disease recurrence 0cschraa bmc cancer page of patients will be censored at the last date of followup if adate of death is not recorded and at the date of death ifthe cause of death is not due to ccquality of lifeqol is measured within the cohort at regular intervalsin patients who gave consent to send questionnaires nationally and internationally validated questionnaires areused among which the european anisation for research and treatment of cancer quality of life questionnaire core and the colorectal cancer moduleeortcqlqc30 and cr29 the work ability indexwai the euro quality of life5 dimensions eq5dthe multidimensional fatigue inventory20 mfi20and the hospital anxiety and depression score hadscosteffectiveness of the ctdnaguided treatmentthe costeffectiveness analysis will be carried out from asocietal perspectiveincluding both direct health carecosts as well as indirect costs from productivity loss thehealth outcome measure in the costeffectiveness analysis will be the total quality adjusted life years qalyper group for analysis offactors related to qalysquestionnaires are used provided within plcrcsample size considerationsthe primary endpoint is the proportion of ctdna positive patients starting with act however 2year rr inthe ctdna positive patients after surgery is an importantsecondary endpoint and the power calculation is performed for this secondary endpoint we estimate thatsimilar to effectiveness in stage iii cc patients act inctdnabased highrisk stage ii cc patients will lead toa absolute reduction of recurrences within yearsafter surgery in the observational trial of patientswith detectable ctdna experienced disease recurrencewithin years after resection with a power of and an alpha of patientswith detectable ctdna need to be included in botharms assuming a prevalence of ctdna after surgery of and adjustment for loss to followup and rejection ofadjuvant therapy in the intervention arm of a totalsample size of patients is calculated in eacharm we expect few patients with detectable ctdna inthe intervention group to refuse act because patientsare selected upfront for being in a physical condition toreceive act and the established prognostic value of detectable ctdna is highwe assume that crossover from the control arm tothe intervention arm will not occur because only eligiblepatients randomly selected in the cohort and allocatedto the intervention arm will be informed about the trialand have the opportunity for immediate analysis ofctdna patients in the control group will not be informed about the trial or their ctdna statuswe assume that of patients in the interventionarm with detectable ctdna will be treated with actthe proportion of patients starting with chemotherapythe primary endpoint can in that instance be determined with a margin of error width of the confidence interval of we expect to complete recruitment of patients within years with more than participating dutchhospitalsdata analysisdata will be analyzed according to the intentiontotreatprinciple for the primary endpoint and the secondaryendpoint of 2year rr in patients with detectable ctdnaafter surgery in this analysis we expect to compare patients with detectable ctdna who received act inthe intervention arm with patients with detectablectdna in the control arm ie based on ctdna analysisperformed retrospectively at least months after surgery and not disclosed to patients and treating physicians the proportion of patients that experience arecurrence in both arms will be compared by means of achisquare test in addition for other secondary endpoints and exploratory analyses we will analyze timetoevent outcomes in patients in both arms with detectablectdna after surgery differences in timetoevent outcomes will be analyzed by standard survival methodseg kaplanmeier curves compared by logrank testscoxs proportional hazards models will be used for multivariable analysiscomparison of qol of the ctdna positive patients inboth study arms will be done using repeated measurements methods and including act as factor qol willalso be analyzed for the whole population in both armsof the study treatment differences at each qol assessment time point will be compared by means of thewilcoxon rank sum testa lifetime horizon will be applied forthe costeffectiveness analysis parametric survival functions willbe used to extrapolate dfs and os curves beyond yearsresponsibilitiesprotocol modifications will be submitted as amendmentto the medical ethical committee by the study coordinator the local principle investigator of each participating hospital is responsible for patient inclusion logisticsof biomaterials to the centrallaboratory and patientfollowup to ensure quality of data study integrity andcompliance with the protocol and the various applicableregulations and guidelines a data monitor of the iknlhas been appointed to conductto thesite visits 0cschraa bmc cancer page of participating centers and randomly check patient datathe study coordinator together with the principle investigator will have access to the final dataset and is responsible for publishing study results the results will besubmitted to a peerreviewed journaldiscussionmedocccreate is the first clinical trial using thetwics design to investigate ctdnaguided strategies instage ii cc taking an important step towards clinicalimplementation of ctdna in cancer diagnostics andcarewithctdnadetectablea few other trials with the aim to reduce recurrencesin cc by use of a ctdnaguided approach are in preparation or recently started the improveit trial adanish study started in october uses a classicalrct in stage i and ii crc patients randomizing between months of act or intensified followup for patientspostsurgerynct03748680 four hundred fifty stage ii crc patients are being included in the australian dynamicstudy and randomized to be treated according to thectdna result with to months of act or accordingto standard of care actrn12615000381583 thecobra study in the united states and canada has asimilar rct approach nrggi also several trialsin stage iii crc patients started recently dynamiciii actrn12617001566325 in the near future thesestudies will provide deeper understanding and lead toimplementation of ctdnaguided strategies in clinicalpracticein the current era of rapidly emerging new diagnosticand treatment strategies the classical rct is challengedbecause of inefficient and therefore timeconsuming recruitment of eligible patients main reasons for patientsto refrain from participation in rcts are preference forone ofthe treatment arms anxiety or aversion torandomization and difficulties understanding the concept of an rct resulting in a delay of availability ofpotential beneficial treatments modern trial designsare being adopted to avoid thistimeconsuming and costly way of conducting trials with highrates of unfinished studies therefore the medocccreate trial uses the modern twics design the twicsdesign has shown to have a positive impact on trialefficiency also by enrolling higher proportions of eligible patients generalizability to daily clinical practiceimproves inefficientthisseveralstudy design hasstrengths firstmedocccreate is nested within the large nationwide plcrc cohort study with currently almost included crc patients the infrastructure of this cohortin which clinical data and biomaterials are collected afterbroad informed consent of participating patients allowsinnovative and efficientcomprehensiveresearch incrc using this infrastructure the study can be quicklyimplemented in many participating hospitals savingcosts and complicated logistics several studies accordingto the twics design are performed within this or comparable cohorts therefore experience with this trialdesign has been gained and this will contribute to execution of the medocccreate study [ ]secondly a difficult ethical dilemma in an rct analyzing ctdna presence postsurgery is avoided by thetwics design with the current knowledge about thestrong association with recurrent disease disclosingctdna status to all participants would be a great burdenfor patients with detectable ctdna and their treatingphysicians in the control group because of disappointment bias in the control group we would expect highdropout and contamination due to crossover when aclassical rct design would be applied making accrualand interpretation of results unfeasible in thistwics study all participants already have blood withdrawn after surgery for research purposes and only theeligible patients allocated to the intervention arm willhave the opportunity to obtain a ctdna test result andact if ctdna is detected patients in the control armtreated according to current guidelines will not be informed about randomization and their blood sampleswill be analyzed at a later point in time beyond the window of act treatmentthis study has also potentiallimitations and challenges the twics design is potentially susceptible tolow statistical power and internal validity biases levelsof participants eligibility and consent should be substantial to achieve valid and reliable results and measurements taken in the control group should be sufficient foradequate comparisons to be made therefore thetwics design is not appropriate for every experimentalintervention in case of the medocccreate studywe argue that eligibility and also consent will be substantial because of the high incidence of cc the large cohortwith high inclusion rates and the assumption that eligible patients in the intervention group are willing toaccept act because of the very strong association of thepresence of ctdna with recurrent diseaseanother limitation is the small sample size for primaryoutcome analysis eventually only patients in botharms of the trial are expected to have detectable ctdnaafter surgery based on previous data relapses areexpected within years and with a high event rate smallnumbers are sufficient capecitabinewe recommend a 6month duration of act consistand oxaliplatin capox oring offluorouracilleucovorin and oxaliplatin folfox forpatients with detectable ctdna after surgery the firstadjuvant cc trials investigating the combination of a 0cschraa bmc cancer page of fluoropyrimidine and oxaliplatin reported results for month duration of act in the idea trialfound a large reduction in toxicity for months treatment compared to months treatment although thistrial could not confirm noninferiority for monthstreatment for all patients treated with capox or folfox in stage iii crc the small difference limits clinicalrelevance besidesit did show noninferiority of theshorter regimen in patients treated with capox consequently dutch guidelines recommend months of actfor cc since however among patients withhighest risk of recurrence t4 n2 or both superiorityof 6month duration of therapy was found additionalideafrance resultsthe esmocongress showed the worst prognosis for ctdnapositive patients who only received months of act therefore in this study we recommend 6monthsact for patients with a very high risk of disease recurrence due to the presence of ctdna after surgerypresentedatliquid biopsy ctdna detection has become a promising technology with multiple putative clinical applications including its potential use as a biomarker for earlydiagnosis prognosis prediction and monitoring of treatment response driven by the excitement of its possibilities the field of technology of ctdna detection andanalysis is rapidly evolving yet the clinical utility ofctdna testing still needs to be proven when to applywhat technology to address which unmet clinical need isa key question that remains to be addressed applying ctdna detection as a biomarker for mrd isa challenging task biologically only a very low amountof ctdna is present in postsurgery patients with mrdstochastically by looking at mutations in a panel ofgenes chances increase that in a given blood sample atleast in one of the genes a mutation can be reliably detected test sensitivity can be further increased by making use of dna mutation information from tumortissue because the stringency in the calling of plasmactdna mutations can be reduced once you know whatmutations to look for tissueinformed ctdna analysisalso increases the ctdna test specificity recent observations showed that ctdna mutation detection can beconfounded by mutations that are present in clonalhematopoiesisincluding mutations in genes that arecommonly affected in cc such as tp53 these confounding mutations can be filtered by applying tissueinformed ctdna analyses as suchtechnically themedocccreate trial makes use of a ctdna test thatis wellsuited for mrd detection clinically howeverthe medocccreate trial needs to resolvewhether a positive ctdna test also allows to select forpatients who truly benefit from act treatment a requirement for clinical implementation to further support clinical implementation of ctdna analyses in thenetherlands the dutch coin initiative aims to providea validation framework for clinicalimplementation ofctdna analyses in the netherlands zonmw projectnumber in conclusion the medocccreate study is thefirst study using the modern and innovative twics design to study ctdnaguided administration of act instage ii cc patients the study aims to answer the important clinical question whether ctdna has prognosticas well as predictive value if this study demonstrates asignificant and substantial difference in disease recurrence in the intervention group compared to the controlgroup ctdna analysis and ctdnaguided treatmentshould be implemented into clinical practice to improvethe prognosis of stage ii cc patientsabbreviationsact adjuvant chemotherapy cc colon cancer cea carcinoembryonicantigen crc colorectal cancer ctdna circulating tumor dnadfs disease free survival dmmr deficient mismatch repair eortcqlqc30 and cr29 european anisation for research and treatment ofcancer quality of life questionnaire c30 and colorectal cancer module eq5d euro quality of life5 dimensions ffpe formalinfixed para | 0 |
immunerelated genes pairs signature predict the prognosis of cervical cancer patientsHan Nie1 Fanqin Bu2 Jiasheng Xu1 Taoshen Li1 Jun Huang2To screen the key immune genes in the development of cervical cancer construct immune related gene pairs IRGPs and evaluate their influence on the prognosis of cervical cancer Tumor Genome Atlas TCGA database and geo database were downloaded as training set and validation set respectively and immune related gene data were downloaded from immport IRGPs model is established by machine learning and the model is analyzed and evaluated Using the Uclcan to analyze the immune genes expression in cervical cancer and to further explore the association with the expression level and the clinical stage and prognosis of cervical cancer According to the analysis of training set we identified IRGPs as key gene pairs and constructed the model The AUC value of the model was greater than and the model group survival rate was conspicuous different P The reliability of the model was confirmed in the validation group Our IRGPs play an important role in the occurrence and development of cervical cancer and can be used as a prognostic marker and potential new target of cervical cancerCervical cancer is one of the four most common gynecological tumors1 Every year at least women in the world are diagnosed with cervical cancer and more than people are killed2 In recent years the incidence rate of cervical cancer has decreased significantly through universal screening and health knowledge However the incidence rate of cervical cancer is still high in developing countries3 For women with low education in less developed areas the coverage rate of cervical cancer screening is still very low4 Squamous cell carcinoma is the most common type of cervical cancer accounting for of cervical cancer cases while adenocarcinoma only accounts for about In developing countries of cervical cancer patients have local infiltration or metastasis which has led to the high mortality of cervical cancer in developing countries Early cervical cancer is usually treated by radical hysterectomy When there are risk factors such as lymph node metastasis and endometriosis that may lead to recurrence they will be treated with chemotherapy5 The standard treatment for patients with locally advanced cervical cancer is conventional radiation therapyCRT6 The fiveyear survival rate of patients with locally advanced cervical cancer can be as high as after surgical resection radiotherapy chemotherapy CRT and so on7 However at present all treatment methods are not effective for patients with paraaortic lymph node metastasis and their threeyear progression free survival time PFS and total survival time OS are and respectively The fiveyear survival rate of cervical cancer patients with recurrence and metastasis was as low as Limited treatment is the main reason for this situation Now palliative chemotherapy is the most commonly used for patients with metastatic and recurrent cervical cancer10 The median survival time of patients with metastatic or recurrent cervical cancer treated with platinumtaxane chemotherapy and bevacizumab can be extended to a0months11 However these treatments are far from enough for most locally advanced and metastatic cervical cancer patients with positive lymph node metastasisIn recent years immunotherapy has been developed and increasingly used in cancer patients For example PDL1 is overexpressed in a variety of tumor cells including liver cancer cells and lung cancer cells and plays an important role in regulating the immune response of tumor cells12 Currently there are several clinical trials involving FDAapproved immunosuppressive checkpoint inhibitors which attack tumor cells expressing PDL1 by blocking the PDL1PD1 signaling pathway so as to improve the treatment and prognosis of patients From the current situation immunosuppressive therapy has achieved good results in many solid tumors16 The 1Department of Vascular Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China 2Department of Gastrointestinal Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China email junhuangncu163comScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cresults of PD1PDL1 inhibition in cervical cancer are also satisfactory However at present immunoassay sites with a therapeutic effect are scarce and the research on tumor immunotherapy is far from sufficient In this study we screened immune genes that are significantly related to the prognosis of cervical cancer constructed an immune gene pair IRGP model based on these genes and used it to verify the unique prognostic markers of cervical cancerMethodData acquisition Gene expression profile data of patients with cervical squamous cell carcinoma were obtained from cancer and tumor gene map TCGA wwwtcga and gene expression profile data set gse4400116 was obtained from gene expression compilation GEO wwwncbinlmnihgovgeo database including samples of cervical cancer patientsAcquisition of sample immune gene expression immune related genes were downloaded from immport wwwimmpo rthome including antigen presenting cells chemokines and their receptors cytokines and their receptors interferon interleukin etc Using limma package in R we compared the gene expression data of cervical cancer samples downloaded from TCGA database and geo database as training set and verification set with immune related genes and extracted the expression amount of immune related genes in cervical cancer samplesConstruction of immune related gene pairs IRGPs In the two groups of data processed in the previous step the IRGP of the sample is calculated and the relatively high change is selected according to the standard of media absolute deviation IRGP values are calculated by comparing gene expression levels in specific samples or profiles in pairs The immune related genes are matched to compare the IRGPs If the first IRG is larger than the second IRG the output of the IRGP is otherwise the output is If the ratio of IRGP score of or in training set and verification set is higher than then remove the IRGP and retain the remaining IRGP as candidate IRGP for prognosis prediction The logistic rank test was used to screen the prognosis IRGP FDR Cox risk regression analysis and glment in R were used to perform tenfold cross validation to analyze the candidate IRGP and obtain the IRGP index We constructed the best gene pairs as immune gene pair model We use ROC to calculate the optimal cutoff value of IRGP index and use it as the basis to distinguish high and low risk groupsIRGPs model validation The single factor and multi factor Cox proportional risk analysis and survival analysis of TCGA and gse44001 cervical cancer samples were carried out with IRGPs modelInfiltration of immune cells in cervical cancer samples In order to study the infiltration of immune cells in the high and low risk groups of cervical cancer we used CIBERSORT17 to evaluate and predict the enrichment of immune cells in the samples CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression RNA SEQ data can be used to estimate the infiltration of immune cells CIBERSORT can analyze the relative abundance of immune infiltrating cells in each sample including NK cells T cells B cells and macrophagesFunctional enrichment analysis of GSEA go and KEGG Gene set enrichment analysisGSEA enrichment analysis was carried out for each gene related to immune prognosis using the fgsea package in R Cluster profiler19 was used to enrich Gene ontologyGOfunction and KEGG pathway Significant enrichment criteria the absolute value of NES is greater than the nomp value is less than and the fdrq value is less than Expression of immune gene in cervical cancer Ualcan were used to analyze the expression of immune genes in cervical cancerStatistical analysis Measured data were expressed as mean ± standard deviation x ± s and data were compared using t test Kaplan Meier method was used for survival analysis The receiver operating characteristic curve ROC curve and ROC analysis were completed by survivalROC103 Single factor and multi factor analysis using Cox proportional risk regression model P was statistically significant P a0 as the difference has very significant statistical significanceEthical approval and consent to participate This article does not contain any studies with patients or animals performed by any of the authorsResultsExpression of immune related genes and construction of IRGPs in cervical cancer samples We obtained gene expression data of cervical cancer samples from TCGA database as training set cervical cancer samples from gse44001 as verification set immune related genes from immport and immune related genes from cervical cancer samples by comparing the two Through these immune related genes we constructed IRGPs We remove more than of the IRGP with a score of or from the training set and validation set leaving IRGP as candidates Combine TCGA clinical data with training set data Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cTCGA clincial dataAge ¥ ¥ GradeG1G2G3G4TT1T2T3T4MM0M1NN0N1Table TCGA clinical dataIRG1APOBEC3HARG2BTCCCL2CCL20CCL20CCL20CCL28CXCL1CXCL2DESDESDLL4FLT3LGHCKHCKHLADQA2IL1BIL1BJAK1NOD1NRP1PLXNB3PSMD7RBP7RBP7STC1TLR3VAV3Immune processesAntimicrobialsAntimicrobialsCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesAntimicrobialsCytokinesAntimicrobialsAntimicrobialsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsAntimicrobialsAntimicrobialsCytokine_ReceptorsCytokine_ReceptorsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsCytokinesAntimicrobialsBCRSignalingPathwayTable Model information about IRGPIImmune processesCytokinesCytokinesCytokinesCytokine_ReceptorsIRG2BTCCLCF1IL16FGFR3APOBEC3C AntimicrobialsARAFPLXNA1MAP3K14TNFSF10PTAFREPORVEGFCDESINHBASAA2STC2LTB4R2DUOX1EDN1APOBEC3C AntimicrobialsCSF2RBCD3DFGFR2SHC1CXCR3DESTNFRSF18CXCR6NRP1NaturalKiller_Cell_CytotoxicityChemokine_ReceptorsTCRsignalingPathwayTNF_Family_MembersChemokine_ReceptorsCytokine_ReceptorsCytokinesCytokinesTGFb_Family_MemberChemokinesCytokinesCytokine_ReceptorsAntimicrobialsChemokinesCytokine_ReceptorsTCRsignalingPathwayCytokine_ReceptorsNaturalKiller_Cell_CytotoxicityChemokine_ReceptorsCytokinesCytokine_ReceptorsAntimicrobialsCytokine_ReceptorsCoefficientScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A Timedependent ROC curve for IRGPI in the training cohort B Timedependent ROC curve for IRGPI in a0year C Timedependent ROC curve for IRGPI in a0year D Timedependent ROC curve for IRGPI in a0yearTable a0 prognosis related IRGPs were screened by lasso Cox proportional risk regression analysis After iterations we selected optimal IRGPs to build the immune prognosis model Table a0IRGPs model validation The immune prognosis model was applied to the training set and the patients in each training set were scored According to ROC curve analysis the optimal cutoff value for patients to be divided into high and low risk groups is Fig a01A After evaluating the model we found that AUC value of model and a0years is Fig a01B Fig a01C and Fig a01D The results show that our immune prognosis gene has a high reliability for the model The training set was divided into highrisk group Fig a02A and highrisk group Fig a02B The results showed that the overall survival rate OS of highrisk group was significantly lower than that of lowrisk group For TCGA training set data single factor and multi factor Cox risk regression analysis showed that only IRGPs model showed significant prognostic effect in single factor Cox Fig a02C while age and IRGPs could be significant independent prognostic factors in multi factor Cox Fig a02D Applying this model to the validation set of gse44001 Fig a03A Table a0 survival analysis showed that the OS of patients in the highrisk group was significantly lower than that in the lowrisk group Fig a03B In the single factor and multi factor Cox analysis IRGPs model and tumor size were significantly correlated with prognosis Fig a03CDInfiltration of immune cells in cervical cancer samples Most studies believe that the occurrence and development of tumor are closely related to immune cells so it is an ideal method to study the infiltration of immune cells in tumor We used CIBERSORT to analyze the infiltration of kinds of immune cells in patients with high and low risk groups Figure a04A shows the expression of immune cells in different risk groups Macrophage M0 Fig a04B activated mast cells Fig a04C were significantly overexpressed in the highrisk group while stationary dendritic cells Fig a04D stationary mast cells Fig a04E activated CD4T cells Fig a04F and cd8t cells Fig a04G were overexpressed in the lowrisk groupScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A The model divides the training set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Training set single factor Cox regression analysis forest map D Training set multivariate Cox regression analysis forest mapFunctional enrichment analysis of GSEA go and KEGG We analyzed the function enrichment of IRGP in the model The results of go analysis showed that IRGP in the model was mainly enriched in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the binding of fibroblast growth factors and the activity of tyrosine kinase Fig a05AB KEGG results showed that these IRGP were involved in cytokine cytokine receptor interaction chemokine signaling tumor necrosis factor signaling MAPK signaling NF kappa B signaling natural killer cellmediated cytotoxicity viral proteins and cytokines and Th1 and Th2 cell differentiation Fig a05CD The results of GSEA Fig a06A showed that these IRGP were significantly enriched in trace ribonucleoprotein complex Fig a06B neurotransmitter transporter activity Fig a06C endopeptidase activity Fig a06D fibroblast growth factor receptor binding Fig a06E hormone activity Fig a06F fibroblast cell proliferation Fig a06G and growth factor receptor binding Fig a06HExpression of immune gene in cervical cancer We explored the expression of IRGP in cervical cancer using the ualcan model Table a0 There were lowlevel expression of IRGP in cervical cancer Fig a0 and highlevel expression of IRGP Fig a0 There were differences in the expression of low expression IRGP and high expression IRGP in different age groups Fig a0 and there were differences in the expression of IRGP in different stages of cervical cancer Fig a0DiscussionCervical cancer is one of the most common gynecological malignancies HPV infection is considered to be the main cause of cervical cancer2122 although the incidence rate of cervical cancer has been significantly decreased due to the development and promotion of HPV vaccine23 But incidence rate of cervical cancer is still high in developing countries and Chinas low income countries24 At present for cervical cancer patients without invasion and lymphatic metastasis the effect of surgery combined with radiotherapy and chemotherapy is better If metastasis and infiltration occur the treatment effect of cervical cancer patients will become very unsatisfactory In recent years immunotherapy has performed well in a variety of cancers including cervical cancer25 Blocking PDL1 PD1 signaling pathway to attack tumor cells expressing PDL1 is the current mainstream method28 Although the anticancer activity of PD1 and PDL1 inhibitors is exciting such immunotherapy is not effective for all patients and a metaanalysis shows that patients who receive PD1 PDL1 inhibitors have a Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A The model divides the validation set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Validation set single factor Cox regression analysis forest map D Validation set multivariate Cox regression analysis forest mapGSE44001 clincial dataStageLargest diameter cm ¥ ¥ ¥ Table GSE44001 clincial datahigher risk of rash thyroid dysfunction pruritus pneumonia and colitis29 Therefore it is of great significance for the detection and treatment of cervical cancer to predict and find more biomarkers that may be related to immune prognosisAt present most of the prognostic genes need to be standardized to reduce the errors caused by sequencing platform and samples In this study the scores of IRGPs constructed by us are calculated from the gene expression data of the same sample which can not only ignore the impact of different platforms but also do not need to standardize and scale the data This method has been used in many studies including cancer molecular classification with high reliability3233In this study we screened pairs of IRGP to construct the immune prognosis model related to the overall survival rate of cervical cancer patients The AUC values of the model in and a0years were all greater than According to these pairs of IRGP they were divided into highrisk group and lowrisk group In TCGA training group and GSE44001 verification group the OS of highrisk group was significantly lower than that of lowrisk group P These pairs of IRGP have a good effect on sample discrimination We found that macrophage Mo and activated mast cells were significantly over expressed in highrisk group by immunocyte infiltration analysis of samples The existing research shows that mast cells and macrophages play an important Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Immune infiltration status within IRGPI risk groups B Expression of Macrophage M0 C Expression of Mast cells activated D Expression of Dendritic cells resting E Expression of Mast cells resting F Expression of T cells CD4 memory activated G T cells CD8role in cervical cancer which can promote the development of cervical cancer by promoting lymphangiogenesis and angiogenesis34 However in the lowrisk group the expression of static dendritic cells static mast cells activated CD4T cells and cd8t cells is high Although the effect of CD4T cells on cervical cancer has not been agreed the cd8t cells are closely related to the better prognosis of cervical cancer patients37 there is evidence that dendritic cells will decrease in patients with high HPV infection which indicates that high expression of dendritic cells is beneficial to resist cervical cancer40 which is consistent with our results The enrichment analysis of go and GSEA showed that these immune genes were mainly involved in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the activity of metalloendopeptidase the binding of fibroblast growth factors and their receptors hormone activity fibroblast proliferation and the binding process of growth Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Histogram graph of Immunerelated genes GO analysis results B Point graph of Immunerelated genes GO analysis results C Histogram graph of Immunerelated genes KEGG pathway analysis results D Point graph of Immunerelated genes KEGG pathway analysis resultsfactor receptorsAs we all know cytokines and chemokines are the key factors in the immune response for example In cervical cancer IL10 can interfere with the differentiation of dendritic cells and thus play a strong immunosuppressive effectTGFβ can inhibit T cell proliferation and attenuate immune response41 Research shows that growth factors and epidermal growth factors are closely related to the growth of cervical cancer and the survival rate of cervical cancer patients High expression of growth factors and epidermal growth factors often predict poor prognosis42 Growth of fibroblasts can stimulate angiogenesis at the early stage of tumor The proliferation and invasion of cancer cells and the remodeling of extracellular matrix promote the growth of cervical cancer4546 KEGG results showed that these immune genes were mainly enriched in chemokine signaling pathway tumor necrosis factor signaling pathway MAPK signaling pathway NF kappa B signaling pathway natural killer cellmediated cytotoxicity viral protein and cytokine and Th1 and Th2 cell differentiation Th1 and Th2 may be involved in the pathogenesis and growth of cervical cancer Th1 may be the target of predicting chemotherapy response of advanced cervical cancer47 while other pathways are classical signal pathways related to cancer Immune cytokines play an important role in cervical lesions Torres et a0al Found that IL10 is highly expressed in the cervix of women with persistent HPV which may be related to the persistence of HPV and the promotion of disease progression Further research by their team showed that copy individuals of IL4 IL6 IL10 and TGFB1 were significantly associated with cervical cancer and could be used as biomarkers for susceptibility to the disease5152These pairs of IRGP have different immune genes most of which are cytokines antimicrobial agents and natural killer cells which are involved in various stimulation reactions and play a key role In cervical cancer HPV can inhibit the apoptosis of cervical cancer cells by down regulating NOD153 In our sample we also found that the expression of NOD1 in tumor tissue is low and there are differences in different ages and stages Figs a07D 9C 10I Sang Yeon Cho et a0al Found that duox1 is highly expressed in cervical squamous cell carcinoma and can play a good prognostic role by increasing the amount of innate immune cells54 The analysis also showed that DUOX1 is highly expressed in tumor tissues and related to age and grade Figs a08B 9G 10D Stc2 can promote the proliferation of cervical cancer cells and increase the resistance to cisplatin55 while high expression of DDL4 is usually associated with low pelvic lymph node metastasis and survival rate of cervical cancer56 Therefore we believe that the IRGP constructed in this study plays an important role in the development and prognosis of cervical cancerThere are also some deficiencies in our research Although we select data samples from two databases for analysis and use more advanced methods to reduce the errors caused by platforms samples etc this is still a retrospective analysis If we can carry out a prospective study or obtain clinical samples and evaluate them with Western blot or immunohistochemistry it will be more convincingScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A GSEA analysis of immune signature genes BH In the high immune risk group of cervical cancer cancer marker genes were abundant P FDR Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cCLCF1DLL4INHBANOD1NRP1RBP7CXCR3DUOX1FGFR3AgeNormalvsAge4160YrsAge2140YrsvsAge4160YrsAge2140YrsvsAge6180YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsAge6180YrsvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsHLADQA2NormalvsAge4160YrsLTB4R2STC2TNFSF10VAV3NormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge4160YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsAge4160YrsvsAge6180YrsPval777E05123E02483E04115E07209E02433E07270E06354E05281E02982E03260E03384E04322E02163E05600E04868E03106E02109E02122E04396E03281E04459E02421E04876E03441E02105E02384E04566E05218E05803E09139E06225E08111E16204E13725E10305E02422E07693E10174E05433E15162E12550E10341E02803E04159E06458E04192E02524E10415E11559E10162E12162E12934E14108E02208E03StageNormalvsStage2NormalvsStage3Stage1vsStage2Stage1vsStage3Stage1vsStage4NormalvsStage1NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage4Stage3vsStageNormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Pval185E06220E04542E03304E02460E04696E07546E03252E03210E02439E02390E03186E02395E02317E02122E03164E03396E04984E03222E02315E06226E02387E07734E03433E01118E04173E05355E04222E02160E05259E09131E09258E04162E12199E12176E05202E04663E12390E05241E04162E12860E10479E09175E04148E02240E04435E05119E04463E02230E13297E09263E07840E04170E02162E12493E12284E12125E04Table P value of IRGPs expression in different ages and stagesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues B Expression of DLL4 in cervical cancer and normal tissues C Expression of INHBA in cervical cancer and normal tissues D Expression of NOD1 in cervical cancer and normal tissues E Expression of NRP1 in cervical cancer and normal tissues F Expression of RBP7 in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CXCR3 in cervical cancer and normal tissues B Expression of DUOX1 in cervical cancer and normal tissues C Expression of FGFR3 in cervical cancer and normal tissues D Expression of HLADQA2 in cervical cancer and normal tissues E Expression of LTB4R2 in cervical cancer and normal tissues F Expression of STC2 in cervical cancer and normal tissues G Expression of TNFSF10 in cervical cancer and normal tissues H Expression of CESC in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different ages B Expression of DLL4 in cervical cancer and normal tissues at different ages C Expression of NOD1 in cervical cancer and normal tissues at different ages D Expression of NRP1 in cervical cancer and normal tissues at different ages E Expression of RBP7 in cervical cancer and normal tissues at different ages F Expression of CXCR3 in cervical cancer and normal tissues at different ages G Expression of DUOX1 in cervical cancer and normal tissues at different ages H Expression of FGFR3 in cervical cancer and normal tissues at different ages I Expression of HLADQA2 in cervical cancer and normal tissues at different ages J Expression of LTB4R2 in cervical cancer and normal tissues at different ages K Expression of STC2 in cervical cancer and normal tissues at different ages L Expression of TNFSF10 in cervical cancer and normal tissues at different ages M Expression of VAV3 in cervical cancer and normal tissues at different agesConclusionWe constructed an immune gene pair model which is closely related to the prognosis of cervical cancer patients The model contains IRGP and immunerelated genes The biological functions of these immunerelated genes are closely related to the occurrence and development of cervical cancer Therefore we think that these IRGPs may be the target of predicting or diagnosing cervical cancer and suggest that immunotherapy can improve the prognosis of cervical cancer patients by regulating these IRGPsScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different stages B Expression of CXCR3 in cervical cancer and normal tissues at different stages C Expression of DLL4 in cervical cancer and normal tissues at different stages D Expression of DUOX1 in cervical cancer and normal tissues at different stages E Expression of FGFR3 in cervical cancer and normal tissues at different stages F Expression of HLADQA2 in cervical cancer and normal tissues at different stages G Expression of INHBA in cervical cancer and normal tissues at different stages H Expression of LTB4R2 in cervical cancer and normal tissues at different stages I Expression of NOD1 in cervical cancer and normal tissues at different stages J Expression of NRP1 in cervical cancer and normal tissues at different stages K Expression of RBP7 in cervical cancer and normal tissues at different stages L Expression of STC2 in cervical cancer and normal tissues at different stages M Expression of TNFSF10 in cervical cancer and normal tissues at different stages N Expression of VAV3 in cervical cancer and normal tissues at different stagesData availabilityAll data are available Please contact us to access if it is neededReceived May Accepted July References Stewart Bernard W et al Cancer prevention as part of precision medicine plenty to be done Carcinogenesis 101093carci nbgv16 Bray F et al Global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin 103322caac21492 Passos Camila M Sales Jacqueline B Maia Emanuella G Caldeira ThaÃs C M Rodrigues Roberta D Figueiredo N Claro Rafael M Trends in access to female cancer screening in Brazil J Public Health Oxf 101093pubme dfdaa0 Asrabuddhe V V Parham G P Mwanahamuntu M H Vermund S H Cervical cancer prevention in low and middleincome countries feasible affordable essential Cancer Prevent Res 10115819406207CAPR110540 Koh WuiJin AbuRustum Nadeem R Bean Sarah Bradley Kristin Campos Susana M Cho Kathleen R Chon Hye Sook Chu Christina Clark Rachel Cohn David Crispens Marta Ann Damast Shari Dorigo Oliver Eifel Patricia J Fisher Christine M Frederick Peter Gaffney David K Han Ernest Huh Warner K Lurain John R Mariani Andrea Mutch David Nagel Christa Nekhlyudov Larissa Fader Amanda Nickles Remmenga Steven W Reynolds R Kevin Tillmanns Todd Ueda Stefanie Wyse Emily Yashar Catheryn M McMillian Nicole R Scavone Jillian L2019 Cervical Cancer Version NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw 106004jnccn Chen J et al Nanotechnology in the management of cervical cancer Rev Med Virol 25Suppl 101002 Varia M A et al Cervical carcinoma metastatic to paraaortic nodes extended field radiation therapy with concomitant 5fluorouracil and cisplatin chemotherapy a Gynecologic Oncology Group study Int J Radiat Oncol Biol Phys 101016s0360 Randall Leslie M Monk Bradley J Darcy Kathleen M Tian C Burger Robert A Liao SY Peters William A Stock Richard J Fruehauf John P Markers of angiogenesis in highrisk earlystage cervical cancer a Gynecologic Oncology Group study Gynecol Oncol 101016jygyno Boussios S et al Management of patients with recurrentadvanced cervical can | 2 |
significant genebiomarkers of primary colorectal cancerJing Han Xue Zhang Yan Liu Li Jing Yibing Liu andDepartment of Medical Oncology The Fourth Hospital of Hebei Medical University Jiankang Road Shijiazhuang Hebei PR ChinaLi FengCorrespondence Li Feng lifeng191gqsinacomBackground Primary colorectal cancer PCRC is a common digestive tract cancer in theelderly However the treatment effect of PCRC is still limited and the longterm survival rateis low Therefore further exploring the pathogenesis of PCRC and searching for specificmolecular targets for diagnosis are the development trends of precise medical treatmentwhich have important clinical significanceMethods The public data were downloaded from Gene Expression Omnibus GEOdatabase Verification for repeatability of intragroup data was performed by Pearsons correlation test and principal component analysis Differentially expressed genes DEGs between normal and PCRC were identified and the proteinprotein interaction PPI networkwas constructed Significant module and hub genes were found in the PPI network A total of PCRC patients were recruited between and from the Fourth Hospital of Hebei Medical University RTPCR was used to measure the relative expression ofCLCA4 and MS4A12 Furthermore the study explored the effect of expression of CLCA4and MS4A12 for overall survivalResults A total of DEGs were identified between PCRC and normal colorectal tissuesTen hub genes concerned to PCRC were screened namely CLCA4 GUCA2A GCG SSTMS4A12 PLP1 CHGA PYY VIP and GUCA2B The PCRC patients with low expressionof CLCA4 and MS4A12 has a worse overall survival than high expression of CLCA4 andMS4A12 P005Conclusion The research of DEGs in PCRC DEGs hub genes especially CLCA4and MS4A12 and related signaling pathways is conducive to the differential analysis of themolecular mechanism of PCRCIntroductionPrimary colorectal cancer PCRC is a common digestive tract cancer in the elderly The primary lesioncan be seen in the left colon the right colon the upper or lower rectum [] PCRC is the second mostcommonly diagnosed cancer in women and the third most commonly diagnosed cancer in men and theprevalence of male is higher than that of female in most areas [] With the social environment lifestyleand dietary structure changes the incidence of PCRC is on the rise and there is a trend of rejuvenationThis is a social issue worthy of attention [] At present there is controversy about the pathogenesis ofPCRC It is generally believed that smoking drinking greasy diet obesity lack of exercise colorectalinflammation and genetic factors are all involved in the onset of cancer But these factors are also the causeof many other tumors Therefore the specific etiological mechanism of PCRC has not yet been elucidated[] Some scholars believe that some genes or molecules are involved in the development of PCRC Thesefindings promote the research and treatment of PCRC [] At present the treatment of PCRC includestraditional surgery chemotherapy radiotherapy emerging immunotherapy molecular targeted therapyetc Clinically the single or combination therapy that best suits the condition is usually selected accordingto the actual situation of the patient [] However the treatment effect of PCRC is still limited andReceived March Revised August Accepted August Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963the longterm survival rate is low The early prognosis of patients with early diagnosis is often better [] Thereforefurther exploring the pathogenesis of PCRC searching for specific molecular targets for diagnosis and treatment realizing early diagnosis targeted treatment and individualized treatment are the development trends of precise medicaltreatment which have important clinical significancePersonalized medicine refers to the treatment of existing diseases based on the information of each persons disease genome [] It is now widely believed that majority of individual differences in drug response are due to geneticfactors Personalized medicine is a discipline that emphasizes studying the effect of genetic factors on a drug [] Recently due to the smooth implementation of the human genome project and the rapid development of bioinformaticspersonalized medicine has been strongly promoted and the concept has been gradually developed []Bioinformatics is a method to process and analyze biological data by combining biological knowledge with information processing technology It is commonly used in highthroughput data analysis such as gene and proteomicsAs a frontier interdisciplinary subject bioinformatics analysis technology can realize the biological analysis of thestructure and function of histological data find the genes or proteins most relevant to diseases and further analysis may find the molecules most relevant to diseases and can be used as disease markers [] At present a largenumber of scholars have applied this technology to tumor research that is processing gene sequence or omics databy bioinformatics analysis technology to find genes or molecular markers most relevant to tumors []Therefore the present study aimed to use the bioinformatics to identify the hub genes of PCRC and to verify theirrole on the overall survival of patients with PCRC based on the clinical data And the research would provide novelinsights for the personalized medicine on the treatment of patients with PCRCMaterial and methodsLease start with dates and time location of study and the recruitmentsof patientsThe present study recruited a total of PCRC patients between and from the Fourth Hospital of HebeiMedical University Shijiazhuang of Hebei province Clinical and histopathological characteristics and followup andsurvival information were available for all patients and were collected retrospectively from medical records Patientsaged years old histologically confirmed as PCRC not received tumor treatment and no history of gastrointestinal surgery will be screened for inclusion criteria Exclusion criteria included age years old or yearsold combined with other malignant tumors operation time more than one month after the last examination andsevers heart diseaseEthical clearance and informed contentThe research conformed to the Declaration of Helsinki and was authorized by the Human Ethics and Research EthicsCommittees of the Fourth Hospital of Hebei Medical University The written informed consents were obtained fromall participatesDownload public dataThe Gene Expression Omnibus GEO database [] httpwwwncbinlmnihgovgeo is the largest most comprehensive and publicly available source of gene expression data It contains information about the expression levels ofmultiple genes in different groups of clinical samples such as the differences in gene expression between tumor tissues and normal tissues GSE41258 GPL96 [HGU133A] Affymetrix Human Genome U133A Array and GSE81558GPL15207 [PrimeView] Affymetrix Human Gene Expression Array were obtained from the GEO database A totalof samples including tumor colorectum tissues from PCRC patients and normal colorectum tissues wereselected from GSE41258 A total of samples including tumor colorectum tissues from PCRC patients and normal colorectum tissues were selected from GSE81558Veriï¬cation for repeatability of intragroup dataFirst repeatability of intragroup data were verified by the Pearsons correlation test The heatmap was drew via the Rlanguage environment and presented the correlation among intragroup data Second principal component analysisPCA was the general method for sample clustering and is commonly performed for diversity analysis resequencinggene expression and other sample clustering based on various variable information The verification for repeatabilityof intragroup data was executed by PCA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Differentially expressed genes DEGs between normal and PCRCGEO2R [] httpwwwncbinlmnihgovgeogeo2r could import data of the GEO database into the R languageand perform differential analysis essentially through the following two R packages including limma packages andGEOquery Therefore through the GEO2R tool DEGs were identified between normal and PCRC group The adjusted Pvalues adj P and the fold change FC ¥ or ¤ were defined as significance SangerBoxshengxinren one tool was used to draw volcano maps [] Venn diagrams were delineated using anonline Venn tool httpbioinformaticspsbugentbewebtoolsVenn which would visualize common DEGs sharedbetween GSE41258 and GSE81558Proteinprotein interaction PPI networkThe common DEGs shared between GSE41258 and GSE81558 were converted into differently expressed proteinsThe STRING Search Tool for the Retrieval of Interacting Genes online database tringdb could construct PPI network which was visualized by Cytoscape version []GO and KEGG analysis via DAVID toolOne online tool DAVID davidncifcrfgovhomejsp version Maryland America was applied to carriedout the functional annotation for DEGs Gene Ontology GO [] generally perform enrichment analysis of genomesAnd there are mainly cellular components CC biological processes BP and molecular functions MF in theGO analysis Kyoto Encyclopedia of Genes and Genomes KEGG wwwkeggjp [] is a comprehensivedatabase of genomic chemical and systemic functional information Therefore DAVID was used to make analysisof GO and KEGGSigniï¬cant module and hub genesMolecular Complex Detection tool MCODE version [] an plugin of Cytoscape was performed toidentify tested most significant module from the PPI network and the criteria was that the maximum depth MCODE scores cutoff kscore and node score cutoff Then cytoHubba [] a free plugin of Cytoscape was applied to authorize the hub genes when the degree ¥ChiaHao Chins [] research introduce a novel Cytoscape plugin cytoHubba for ranking nodes in a network by theirnetwork features CytoHubba provide a userfriendly interface to explore important nodes in biological networksWhen the degree¥ in the cytoHubba the hub genes would be obtained And in the former publications []numerous researchers chose hub genes out of the DEGs Therefore the present study chose hub genes out of DEGsInteraction between the hub genesPearsons correlation analysis was also performed to present the interaction between the hub genes The cBioPortalhttpwwwcbioportal [] one online software constructed the coexpression network of these hub genesSimultaneously the coexpression network of hub genes in the field of PCRC was also analyzed via Coexpedia a freeand online toolhttpwwwcoexpedia []Expression analysis of hub genesUCSC Xena xenaucsceduwelcometoucscxena could integrate the public genomic data sets to analyzeand visualize the expression level of hub genes Then the clustering analysis of expression level of hub genes wasperformed using heatmaps based on the GSE41258 and GSE81558 Also the expression profiles of hub genes in thehuman different ans were displayed with Gene Expression Profiling Interactive Analysis GEPIA httpgepiacancerpkucn [] In order to compare the expression of hub genes in the various tumors GEPIA was used Andthe expression profiles of hub genes in the PCRC and normal groups were analyzed using GEPIAEffect of hub gene expression for pathological stage and overall survivalEffect of hub gene expression for pathological stage and overall survival was analyzed by the GEPIA Finally the correlation and linear regression analysis between PCRC and hub gene expression were performed And the receiveroperator characteristic ROC curve analysis was performed to test the sensitivity and specificity of hub gene expression for diagnose PCRC The SPSS software version IBM New York America was used to conduct all thestatistical analysis A Pvalue was defined as statistical significance The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table Summaries for the function of hub genesNoGene symbolFull nameFunctionCLCA4GUCA2AChloride channel accessory Guanylate cyclase activator 2AGCGSSTGlucagonSomatostatinMay be involved in mediating calciumactivated chloride conductanceEndogenous activator of intestinal guanylate cyclase It stimulates this enzyme through thesame receptor binding region as the heatstable enterotoxinsRegulates blood glucose by increasing gluconeogenesis and decreasing glycolysisGLP1 is a potent stimulator of glucosedependent insulin release GLP2 stimulatesintestinal growth concomitant with increased crypt cell proliferationSomatostatin inhibits the release of somatotropin This hormone is an important regulatorof the endocrine system through its interactions with pituitary growth hormone thyroidstimulating hormone and most hormones of the gastrointestinal tractMS4A12Membrane spanning 4domainsMay be involved in signal transduction as a component of a multimeric receptor complexA12Silencing of this gene in colon cancer cells inhibits the proliferation cell motility andchemotactic invasion of cellsPLP1CHGAPYYVIPProteolipid protein This is the major myelin protein from the central nervous system It plays an important rolein the formation or maintenance of the multilamellar structure of myelinChromogranin AThis gene product is a precursor to three biologically active peptides vasostatinpancreastatin and parastatinPeptide YYThis gut peptide inhibits exocrine pancreatic secretion has a vasoconstrictory action andinhibitis jejunal and colonic mobilityVasoactive intestinal peptideVIP causes vasodilation lowers arterial blood pressure stimulates myocardial contractilityincreases glycogenolysis and relaxes the smooth muscle of trachea stomach andgallbladderGUCA2BGuanylate cyclase activator 2B May be a potent physiological regulator of intestinal ï¬uid and electrolyte transport May bean autocrineparacrine regulator of intestinal salt and water transportRTqPCR assayTotal RNA was extracted from tumor colorectum tissues from PCRC patients and adjacent normal colorectum tissuesby the RNAiso Plus Trizol kit Thermofisher Massachusetts America and reverse transcribed to cDNA RTqPCRwas performed using a Light Cycler® System with specific primers for the ten hub genes Table presents theprimer sequences used in the experiments The RQ values 01 01Ct where Ct is the threshold cycle of each samplewere calculated and are presented as fold change in gene expression relative to the control group GAPDH was usedas an endogenous control The expression level of CLCA4 and MS4A12 in PCRC patients was measured by RTqPCROverall survival analysis of the PCRCThe KaplanMeier method was performed to analyze the overall survival All statistical analyses were conductedusing SPSS software version and P005 was considered statistically significantResultsHigh repeatability of dataThere exist strong correlations among samples in the PCRC group and also strong correlations among samples in thecontrol group in the GSE41258 via the Pearsons correlation test Supplementary Figure S1A And there also existstrong correlations among samples in the PCRC group and also strong correlations among samples in the controlgroup in the GSE81558 via the Pearsons correlation test Supplementary Figure S1B Furthermore PCA was performed to verify the repeatability of data Through the PCA the repeatability of the data in GSE41258 was fine Thedistances between per samples in the PCRC group were close and the distances between per samples in the controlgroup were also close in the dimension of PC1 Supplementary Figure S1C Through the PCA the repeatability ofthe data in GSE81558 was fine The distances between per samples in the PCRC group were close and the distancesbetween per samples in the control group were also close in the dimension of PC1 Supplementary Figure S1DDEGs between control and PCRCThere are plenty of DEGs on the all chromosomes between PCRC and control samples Supplementary Figure S1EOne volcano plot presents the DEGs in the GSE41258 Figure 1A and another volcano plot presents the DEGs in theGSE81558 Figure 1B In the volcano plots the green nodes indicate the downregulated DEGs and the red nodesindicate the upregulated DEGs The Venn diagram manifested that a total of DEGs were exist in the two datasetsGSE41258 and GSE81558 simultaneously Figure 1C After construction of PPI network for the common DEGsthere are nodes and edges in the PPI network Figure 1D The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The differently expressed genes and PPI networkA One volcano plot presents the DEGs in the GSE41258 B Another volcano plot presents the DEGs in the GSE81558 In thevolcano plots the green nodes indicate the downregulated DEGs and the red nodes indicate the upregulated DEGs C TheVenn diagram manifested that a total of DEGs were exist in the two datasets GSE41258 and GSE81558 simultaneously DThe PPI network of the common DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963The functional enrichment analysis of DEGs via GO and KEGGGO analysis manifested that variations in DEGs related with biological processes BP were significantly enrichedin bicarbonate transport onecarbon metabolic process cell surface receptor signaling pathway collagen catabolicprocess transport xenobiotic transport body fluid secretion axon development positive regulation of guanylate cyclase activity drug transmembrane transport response to steroid hormone response to tumor necrosis factor positiveregulation of peptidylthreonine phosphorylation cell proliferation and regulation of intracellular pH Figure 2AThe variations in DEGs related with cellular components CC were significantly enriched in extracellular space extracellular region anchored component of membrane proteinaceous extracellular matrix plasma membrane apicalplasma membrane integral component of plasma membrane apical part of cell extracellular exosome and basolateralplasma membrane Figure 2B The variations in DEGs related with molecular functions MF were significantly enriched in hormone activity carbonate dehydratase activity xenobiotictransporting ATPase activity arylesterase activity metalloendopeptidase activity neuropeptide hormone activity and hydrolase activity hydrolyzing Oglycosylcompounds Figure 2C KEGG pathway enrichment analysis showed that the top pathways related with DEGs werenitrogen metabolism bile secretion proximal tubule bicarbonate reclamation and pancreatic secretion Figure 2DSigniï¬cant module network and identiï¬cation of hub genesA significant module was screened from the PPI network and the module network consisted of nodes and edgesFigure 2E And ten hub genes were identified including CLCA4 GUCA2A GCG SST MS4A12 PLP1 CHGAPYY VIP and GUCA2B Figure 2F The function of hub genes were summarized in the Table Strong interaction among the hub genesThrough the Pearsons correlation test heat maps manifested that there were strong correlations among hub genes inthe GSE41258 Supplementary Figure S2A and GSE81558 Supplementary Figure S2B datasets PYY SST GCG andVIP existed simultaneously in the coexpression network via cBioPortal Supplementary Figure S2C And throughthe analysis of Coexpedia there were strong interactions among PYY SST GCG CHGA CLCA4 GUCA2B andMS4A12 Supplementary Figure S2DDifference of expression of hub genes between PCRC and controlsamplesHeat map showed that the expressions of all the hub genes were lower in the PCRC samples than the control samplesSupplementary Figure S2E Hierarchical clustering allowed for simple differentiation of PCRC tissues from normalcolorectal tissues via the expression levels of hub genes in the GSE41258 Supplementary Figure S3A and GSE81558Supplementary Figure S3B datasets The expressions of all the hub genes were lower in the PCRC group than thecontrol groupThe analysis of expression level of hub genesThe hub genes in the human different ans were expressed in the Supplementary Figure S3C The pink presents thetumor individuals and the green presents the normal individuals The expression of hub genes in the colorectum washigher in the normal individuals compared with the tumor samples Supplementary Figure S3C When we comparedthe expression of hub genes in the various tumors the all hub genes were downregulated in the PCRC samples alsonamed colon adenocarcinoma COAD Supplementary Figure S3D Through GEPIA analysis the expressions ofhub genes in the PCRC patients were lower than the normal individuals Supplementary Figure S4AAssociation between hub gene expression pathological stage andoverall survivalThe results of GEPIA manifested that the expression of VIP was significantly positively related with pathological stageP0027 while the expression of CLCA4 GUCA2A GCG SST MS4A12 PLP1 CHGA PYY and GUCA2B wasnot as Supplementary Figure S4B KaplanMeier analysis showed that PCRC patients with low expression levelsof CLCA4 and MS4A12 had poorer overall survival times than those with high expression levels P005 Figure3AE PCRC patients with high expression levels of GCG SST PLP1 and CHGA had poorer overall survival timesthan those with low expression levels P005 Figure 3CDF Supplementary Figure S5A However there was nostatistically significant effect on OS associated with the expression of GUCA2A PYY VIP and GUCA2B P005Figure 3B Supplementary Figure S5BD The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The enrichment analysis for DEGs and the identiï¬cation of hub genesA Detailed information relating to changes in the biological processes BP of DEGs in PCRC and control colorectal samplesB Detailed information relating to changes in the cellular components CC of DEGs in PCRC and control colorectal samples CDetailed information relating to changes in the molecular functions MF of DEGs in PCRC and control colorectal samples D KEGGpathway analysis for DEGs E The significant module of the PPI network F The hub genes identified from the PPI network The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure The overall survival KaplanMeier of six hub genesA CLCA4 B GUCA2A C GCG D SST E MS4A12 F PLP1 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table The correlation and linear regression analysis between PCRC and relevant gene expressionPCRCMultiple linear regressionVIFODTGene symbolPearsons correlation coefï¬cientPvalueÏaCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2BPvalueaPearsons correlation coefï¬cient between PCRC and relevant characteristics Ï Pearsons correlation coefï¬cientbMultiple linear regression analysis PCRC primary colorectal cancer P005 P001 P0001Table Receiver operator characteristic curve analysis of hub gene expression for PCRCGene symbolPCRCCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2BAUC0987maxPvalue95CIAUC area under curve max the maximum of AUC Signiï¬cant variables ODT Optimal diagnostic thresholdPCRC primary colorectal cancer P0001Correlation linear regression and ROC analysisThe Pearsons correlation coefficient was used in the correlation analysis and CLCA4 Ï P0001GUCA2A Ï P0001 GCG Ï P0001 SST Ï P0001 MS4A12 Ï P0001 PLP1 Ï P0001 CHGA Ï P0001 PYY Ï P0001 VIP Ï P0001 and GUCA2B Ï P0001 were significantly correlated with PCRC Table In themultivariate linear regression model holding all other variables at a fixed value the natural logarithmic DN remainedassociated with CLCA4 GUCA2A SST MS4A12 PLP1 CHGA PYY and GUCA2B P005 Table To identify accurate thresholds for hub genes to predict PCRC we constructed ROC The expression of all hubgenes was associated with a diagnosis of PCRC AUC Pvalue0001 Table and Figure The ROCcurve of CLCA4 was shown in Figure 4A and the area under curve of CLCA4 was maximal The ROC curve ofGUCA2A was shown in Figure 4B The ROC curve of GCG was shown in Figure 4C The ROC curve of SST wasshown in Figure 4D The ROC curve of MS4A12 was shown in Figure 4E The ROC curve of PLP1 was shown inFigure 4F The ROC curve of CHGA was shown in Figure 4G The ROC curve of PYY was shown in Figure 4H TheROC curve of VIP was shown in Figure 4I The ROC curve of GUCA2B was shown in Figure 4J The ROC curves ofper hub genes are shown in Figure 4K The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Figure ROC curves of hub genes for PCRCA CLCA4 B GUCA2A C GCG D SST E MS4A12 F PLP1 G CHGA H PYY I VIP J GUCA2B K ROC curves of allhub genes The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963Table Clinicopathological variables and the expression status of CLCA4 and MS4A12CLCA4PMS4A12PLow High Low High SexAgeMaleFemale years¥ yearsOverall survival months¥ monthsPearsons chisquared test was usedP005Figure The veriï¬cation of expression and overall survival analysis for CLCA4 and MS4A12A The relative expression of CLCA4 based on PCR B The relative expression of MS4A12 based on PCR C The overall survivalof PCRC based the expression of CLCA4 D The overall survival of PCRC based the expression of MS4A12Basic information of PCRC patientsPatients basic information were presented in Table The mean patient age was years old range yearsand the median OS was months range monthsRTqPCR analysis validation of hub genesAs presented in the result CLCA4 P005 Figure 5A and MS4A12 P005 Figure 5B were markedly The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20200963101042BSR20200963downregulated in PCRC samples when compared with the adjacent normal colorectum tissues It should be notedthat the expression situation of CLCA4 and MS4A12 were consistent in above results of bioinformaticsLow expression of CLCA4 and MS4A12 in PCRC patients wereindependent prognostic factors for the poor overall survivalThe KaplanMeier OS curves were analyzed Low expression of CLCA4 was predictive of a shorter OS in the PCRC patients P005 Figure 5C Low expression of MS4A12 was predictive of a shorter OS in the PCRC patients P005Figure 5DDiscussionPCRC is a common digestive tract cancer which seriously affects the life expectancy and quality of life of patients Inrecent years the survey results show that the morbidity and mortality rate are on the rise [] The clinical manifestations of patients with PCRC are related to the location and pathological type of the tumor The most common typeof pathology is adenocarcinoma The primary lesion located in the colon often causes diarrhea obstruction bleedingin the rectum anemia and cachexia in the later stage of cancer patients [] The current treatment is mainly surgerycombined with chemotherapy or radiotherapy while advocated exercise to enhance the bodys immunity and preventinfection [] Gavrilas et al found that combination of dietary preparations such as curcumin and resveratrol withchemotherapeutic drugs contributed to the prognosis of PCRC [] Clinical application benefit and safety of epidermal growth factor EGFRrelated targeted therapy and PD1PDL1 immunotherapy are still to be further studied[] The investigation found that the cost of PCRC treatment is high and it takes up a lot of medical resources andthe prognosis of patients is not necessarily proportional to the input The early treatment of early treatment patientshas a relatively low total cost of treatment and a good prognosis [] Therefore to further explore the pathogenesisof PCRC to find possible therapeutic targets to achieve early diagnosis targeted therapy individualized treatmenthas important clinical value and market prospectsBioinformatics has been widely used as a new means of exploring disease mechanism and searching fordiseaserelated genetic molecules Zhang et al found genes related to hepatocellular carcinoma by bioinformaticsanalysis Further analysis confirmed the correlation between these differential genes and diseases suggesting thatthese molecules may be used as molecular targets for early diagnosis and treatment [] Zhang et al found the mostrelevant molecules of gastric cancer miR19b3p and miR165p by analyzing the genomewide miRNA microarraydata of gastric cancer patients which provided a new idea for the diagnosis and treatment of gastric cancer [] Sunfound molecules related to the pathogenesis of colorectal cancer by screening from public databases Further analysisshowed that differentially expressed genes such as PPBP CCL28 and CXCL12 are likely to be involved in the development of colorectal cancer and may be potential diagnostic and therapeutic targets [] We found genes thatwere differentially expressed in patients with PCRC by bioinformatics analysis Low expression of PLP1 VIP SSTGCG PYY MS4A12 CLCA4 GUCA2A CHGA and GUCA2B in tumor patients compared with normal subjectsAt the same time we performed survival analysis on patients with PCRC The results showed that CLCA4 GUCA2AGCG SST MS4A12 and PLP1 genes were significantly associated with the survival of patients with PCRCCLCA4 is the chloride channel accessory CLCA4 is a member of the calciumsensitive chloridetransportingprotein family involved in intracellular ion channel activity chloride ion transmembrane transport and proteolysis Members of the calcium activated chloride channel CLCA gene family are thought to have multiple functionsincluding cell adhesion and tumor suppression Ye et al found that CLCA4 is low expressed in patients with oraltongue squamous cell carcinoma through genomewide transcriptional mapping which provides ideas for diagnosisand targeted therapy [] Bundela also found multiple differentially expressed genes in oral cancer patients in Indiaand suggested that CLCA4 may be a potential therapeutic target [] Yu et al found that CLCA4 is low expressedin breast cancer patients Further analysis revealed that CLCA4 is a marker of breast epithelial differentiation andmay be involved in tumor proliferation and metastasis Clinical data analysis showed that patients with breast cancerwith low expression of CLCA4 had lower recurrencefree survival rate suggesting that it may serve as a diagnosticand therapeutic target [] Hu found that CLCA4 was low expressed in bladder cancer tissues Further analysis revealed that CLCA4 may be involved in the proliferation and invasion of bladder cancer through PI3KAKT signaltransduction suggesting that CLCA4 may be a target for diagnosis and treatment [] Liu found that CLCA4 mayinhibit epithelialmesenchymal transition EMT by affecting PI3KATK phosphorylation thereby inhibiting cellmigration and invasion of hepatoma cells [] Yang found that patients with colorectal cancer CRC had low expression of CLCA1 and CLCA4 and further experiments confirmed that CLCA1 is involv | 2 |
"coronavirus disease COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 emerged in China Currently it is breaking out globally and posing a serious threat to public health The typically clinical characteristics of COVID19 patients were fever and respiratory symptoms and a proportion of patients were accompanied by extrapulmonary symptoms including cardiac injury kidney injury liver injury digestive tract injury and neurological symptoms Angiotensin converting enzyme ACE2 has been proven to be a major receptor for SARSCoV2 and could mediate virus entry into cells And transmembrane protease serine TMPRSS2 could cleave the spike S protein of SARSCoV2 which facilitates the fusion of SARSCoV2 and cellular membranes The mRNA expressions of both ACE2 and TMPRSS2 were observed in the heart digestive tract liver kidney brain and other ans SARSCoV2 may have a capacity to infect extrapulmonary ans due to the expressions of ACE2 and TMPRSS2 in the cells and tissues of these ans It seems that there is a potential involvement of ACE2 and TMPRSS2 expressions in the virus infection of extrapulmonary ans and the manifestation of symptoms related to these ans in patients with COVID19 Here we revealed the expressions of ACE2 and TMPRSS2 in extrapulmonary ans and we also summarized the clinical manifestation and the management of extrapulmonary complications in patients with COVID19 Introduction Since the late a novel coronavirus officially named as severe acute respiratory syndrome coronavirus SARSCoV2 was identified as the pathogen to cause pneumonia [] As a member of the Betacoronavirus genus SARSCoV2 has genomic nucleotides identity with human severe acute respiratory syndrome coronavirus SARSCoV and shares amino acid sequence identity with SARSCoV [] The World Health anization WHO named the disease caused by SARSCoV2 as coronavirus disease COVID19 Until April the virus has swept through countries more than million cases with COVID19 have been confirmed and more than cases died which has been posing significant threats to public health SARSCoV2 can cause respiratory diseases and may lead to acute respiratory distress syndrome ARDS multiple an failure and even death in severe cases [] In addition to typical symptoms such as cough and fever some patients developed the symptoms in multiple systems such as cardiovascular system digestive system and Abbreviations ACE2 Angiotensin converting enzyme AKI Acute kidney injury ALI Acute liver injury ALP Alkaline phosphatase ALS Artificial liver system ALT Alanine aminotransferase AMI Acute myocardial infarction ARDS Acute respiratory distress syndrome AST Aspartate aminotransferase AT2 Alveolar cells BUN Blood urea nitrogen CCLE Cancer Cell Line Encyclopedia CNS Central nervous system COVID19 Coronavirus disease GEO Gene Expression Omnibus GGT Gammaglutamyltransferase GI Gastrointestinal injury GTEx GenotypeTissue Expression ICU Intensive care unit MCS Mechanical circulatory support NP Nucleoprotein PCI Percutaneous coronary intervention SARSCoV2 Severe acute respiratory syndrome coronavirus Scr Serum creatinine ScRNASeq Single cell RNA sequencing STEMI STelevation myocardial infarction TBIL Total bilirubin TEM Transmission electronic microscope TMPRSS2 Transmembrane protease serine VV venousvenous WHO World Health anization Corresponding authors at Department of Infectious Disease and Institute of Hepatology Qingdao Municipal Hospital Qingdao University Digestive Disease Key Laboratory of Qingdao Qingdao China Email addresses xinyongning9812163com Y Xin zlk0823163com L Zhuang 101016jbiopha2020110678 Received June Received in revised form August Accepted August BiomedicinePharmacotherapy1312020110678Availableonline24August2020075333222020TheAuthorsPublishedbyElsevierMassonSASThisisan accessundertheCCBYNCNDlicensehttpcreativecommonslicensesbyncnd40 0cexpressed not only in the cells and tissues of lung but also in extrapulmonary ans [] Fig In this section the expression levels of ACE2 and TMPRSS2 in extrapulmonary ans including heart kidney liver digestive tract brain and other ans were reviewed Heart M Dong nervous system in the early stages of COVID19 which brings more challenges to the timely diagnosis of patients [] Angiotensin converting enzyme ACE2 as a metalloproteinase is a carboxyterminal dipeptidyl peptidase [] The primary physiological role of ACE2 is involved in the regulation of vasoconstriction and blood pressure [] Transmembrane protease serine type2 TMPRSS2 belonging to the type II transmembrane serine protease family could cleave the coronavirus spike S protein [] It was demonstrated that ACE2 and TMPRSS2 were crucial for the entry of SARSCoV and SARSCoV2 into the host cells [] Cell entry of SARSCoV2 depends on binding of the S protein to the specific cellular receptor and S protein priming by host cell proteases As shown in Fig each S protein of SARSCoV2 consists of two subunits a globular S1 domain at the Nterminal region and the membraneproximal S2 domain SARSCoV2 utilizes receptorbinding domain within the S1 domain to bind to the cellular receptor ACE2 which could trigger the effects of TMPRSS2 on the cleavage of protein S at the S1 and S2 sites and priming cell membrane fusion for viral entry [] As receptors and mediators of virus entry are important for determining viral host and an the route of SARSCoV2 infection and the infected an may depend on the expression and distribution of ACE2 and TMPRSS2 [] Studies have shown that ACE2 and TMPRSS2 are expressed not only in lung tissues but also in extrapulmonary ans including heart kidney liver colon esophagus brain gallbladder and testis suggesting that SARSCoV2 may also affect extrapulmonary ans [] In this review the distributions of ACE2 and TMPRSS2 in extrapulmonary ans and the characteristics and clinical managements of extrapulmonary an injury caused by SARSCoV2 were summarized We believe that this will be important in understanding on the infection of extrapulmonary ans in patients with COVID19 The mRNA expressions of ACE2 and TMPRSS2 in extrapulmonary ans The mRNA expressions of ACE2 in different human ans were analyzed and the results showed that ACE2 was expressed in the heart [] Furthermore Chen analyzed the feature of ACE2 expressions among cardiac cell types and found that ACE2 was specifically expressed in pericyte [] Moreover RNA sequencing from patients with failing hearts and normal donors revealed that myocardial ACE2 expressions were significantly increased in patients with heart failure which was further validated at the protein level by proteomics profiling from heart failure and normal donors [] Another study also showed that the expression of ACE2 in heart tissues of patients with underlying heart disease was higher than that in normal heart tissues [] These two studies suggested that the expression of ACE2 in heart tissue of patients with underlying heart disease was higher than that in normal heart tissue Guo et al analyzed the mRNA expression of TMPRSS2 from the GenotypeTissue Expression GTEx database and the results showed that TMPRSS2 is also expressed in the heart [] By singlecell RNA sequencing scRNASeq to profile the gene expression landscapes of cardiac cells from human embryos Qi revealed that the cardiomyocytes from the heart contain ACE2expressed cells and TMPRSS2expressed cells and the cardiovascular progenitor cells and cells TMPRSS2expressed cells respectively [] These data showed that both ACE2 and TMPRSS2 were expressed in the heart contain ACE2expressed Kidney Studying the viral susceptibility of extrapulmonary ans is important for a deeper understanding for the pathogenesis of SARSCoV infection Studies have shown that ACE2 and TMPRSS2 were Expression analysis from the GTEx database showed that kidney displayed the fifth high expression of ACE2 [] To investigate the expression of ACE2 in kidney Lin analyzed the public singlecell transcriptome dataset of normal kidneys from healthy donors the Fig Entry of SARSCoV2 into host cells SARSCoV2 infected the host cells by the spike protein of the virus and the functions of ACE2 and TMPRSS2 in host cells BiomedicinePharmacotherapy13120201106782 0cM Dong Fig Tissue distributions of ACE2 and TMPRSS2 in human AB the schematic diagram of the expressions of ACE2 A and TMPRSS2 B in multiple human tissues The colour strength is corresponding to the gene expression level ACE2 and TMPRSS2 were expressed in the brain and heart ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in the hepatocytes and cholangiocytes ACE2 and TMPRSS2 were highly expressed in kidney and intestinal epithelial cells Both ACE2 and TMPRSS2 were also expressed in the esophagus stomach nose testis pancreas breast prostate and thyroid results showed that the ACE2 was distributed across multiple cell types and was mostly enriched in proximal tubule cells [] Fan et al confirmed the specific ACE2 expression in tubular cells from the Gene Expression Omnibus GEO dataset while it was not observed in immune cells and glomerular parietal epithelial cells RNA and protein expression data of ACE2 in different human tissues and cancer cell lines were obtained from three online datasets including the Cancer Cell Line Encyclopedia CCLE GTEx database and the Human Protein Atlas dataset and the results indicated that both mRNA and protein expression levels of ACE2 were relatively high in kidney cells especially in renal tubular cells [] Meanwhile Suryawanshi analyzed the data of kidney tissues in scRNASeq datasets and found that either proximal tubular cells or tubular progenitor cells in the kidney coexpressed ACE2 and TMPRSS2 [] The data of the scRNAseq from GEO dataset GSE134355 showed that ACE2 and TMPRSS2 expression levels were high in nephron epithelial cells epithelial cells endothelial cells and mesangial cells of the kidney [] Recently Pan also found that the TMPRSS2 gene was coexpressed with ACE2 in kidney podocytes [] These data showed that both ACE2 and TMPRSS2 were highly expressed in tissues and cells of kidney Liver Chai et al analyzed the scRNAseq data from GEO database GSE124395 to evaluate ACE2 gene expression in liver the results showed that ACE2 was highly expressed in cholangiocytes which level was about times higher than that in hepatocytes [] The GTEx database also showed that both ACE2 and TMPRSS2 were expressed in the liver [] Zhou identified that TMPRSS2 is highly expressed in hepatocytes from Human Cell Atlas database [] Recently Wen indicated that ACE2 and TMPRSS2 are specifically coexpression in TROP2 liver progenitors of human liver tissue using scRNA sequencing [] These data indicate that ACE2 expression is expressed at a relative low level in hepatocytes and mainly located in cholangiocytes while TMPRSS2 is expressed in hepatocytes Digestive tract A previous study showed that ACE2 could be found in the upper esophagus and it could be detected in stratified epithelial cells and absorptive enterocytes of the ileum and colon [] Quantitative mRNA expression profiling of ACE2 across human tissues by Harmer showed that ACE2 was expressed at a high level in gastrointestinal tissues [] Zhang et al analyzed datasets with singlecell transcriptomes of esophagus gastric ileum colon and lung and the data showed that ACE2 was not only highly expressed in the type II alveolar cells AT2 of lung but also in the stratified epithelial cells ileum absorptive enterocytes cells and colon enterocytes [] Similarly the immunofluorescent staining of esophagus stomach duodenum and rectum showed that ACE2 was stained mainly in the cytoplasm of gastrointestinal epithelial cells [] Besides the scRNAseq data showed that ACE2 was significantly elevated in the proximal and distal enterocytes [] Guo et al suggested that TMPRSS2 was highly expressed in almost all ans of the digestive tract including colon stomach small intestine and esophagus [] Using published scRNAseq data and seven inhouse normal colon samples Lee reported that the coexpressions of ACE2 and TMPRSS2 transcripts were mainly observed in the small intestine and colon [] The highest expressions of TMPRSS2 and ACE2 were found in enterocytes among the intestinal cell types []These data showed that TMPRSS2 and AEC2 are highly expressed in the digestive tract Nervous system Analysis using the GTEx database showed that both TMPRSS2 and ACE2 are expressed at relatively low levels in the brain cortex [] Chen found that ACE2 was relatively highly expressed in some important brain areas such as the substantia nigra and brain ventricles using seven brain transcriptome databases [] ACE2 was expressed at high level in the piriform cortex of human brain and its expression could also be detected in many neurons including both excitatory and inhibitory neurons and some nonneuron cells including astrocytes and oligodendrocytes in human middle temporal gyrus and posterior cingulate cortex [] Qi analyzed the scRNAseq data of substantia nigra BiomedicinePharmacotherapy13120201106783 0cClinical classification of acute cardiac injury NonICUcases ICU cases NonICUcases ICUcases Nonsevere cases Severecases1965 Recoveredcases Died cases NonICUcases ICUcases Survivor cases Nonsurvivor cases Chen [] Hong [] Zhou [] China Korea China M Dong and cortex of brain from GEO database the results showed that both ACE2 and TMPRSS2 were expressed in the oligodendrocyte precursor cells and the astrocytes of the substantia nigra and cortex [] There are limited reports on the expressions of ACE2 and TMPRSS2 in peripheral nervous system Brann analyzed the ACE2 and TMPRSS2 expression in different cell type from human scRNAseq dataset GSE139522 and found that neither olfactory sensory neurons nor olfactory bulb neurons expressed these two genes while ACE2 and TMPRSS2 were expressed in the nonneuronal cells including the sustentacular cells and olfactory bulb pericytes [] These data showed that ACE2 and TMPRSS2 could also be coexpressed in the nervous system Other ans or tissues Table Characteristics of acute cardiac injury after SARScid0 CoV2 infection Study Basic heart disease Acute cardiac injury Country Subject China Wang [] China NA Huang [] Li [] China Moreover ACE2 and TMPRSS2 were also reported to be coexpressed in some other ans [] It has been revealed that both ACE2 and TMPRSS2 are expressed in testis by scRNA sequencing and expression profile analysis indicating that testicular cells might be the potential targets of SARSCoV2 Another report revealed that multiple kinds of cells in the nose including nasal brushing epithelial cells nasal turbinate epithelial cells and nasal airway epithelial cells contained ACE2expressed and TMPRSS2expressed cell clusters [] Moreover ACE2 and TMPRSS2 were also expressed in pancreas breast prostate and thyroid [] and these ans might also be the targets of SARSCov2 Infection of SARSCoV2 and extrapulmonary an injury of patients with COVID19 SARSCoV2 infection and cardiac injury Recently autopsy analysis by Fox revealed that the histopathology of the heart was consistent with the typical pattern of viral myocarditis [] SARSCoV2 RNA was detected in the cardiac tissues of the patients with COVID19 [] These data suggested that SARSCoV2 may directly infect heart The epidemiology of COVID19 reported that cardiac injury was one of the most severe an damages [] The clinical manifestations of cardiac injury in COVID19 patients are complex and could present with heart failure arrhythmias or acute myocardial infarction AMI [ ] Inciardi reported the first case who had the symptom of heart failure at first and later the patient was positive for SARSCoV2 using nucleic acid test [] Cardiac injury is a common symptom in patients with COVID19 Shi reported that patients with COVID19 had cardiac injury [] Moreover there were patients with acute cardiac injury in a cohort including COVID19 patients and of patients with acute cardiac injury in the intensive care unit ICU [] Furthermore a study by Wang showed that there were patients with acute cardiac injury and patients presented with arrhythmia of COVID19 patients while acute cardiac injury was observed in of patients with CIVID19 in the ICU [] These cases suggested that SARSCoV2 may cause serious heart damage which should be widespreadly concerned Furthermore acute cardiac injury is more prevalent in severe cases with COVID19 [] Table And it has been reported that COVID19 patients with cardiac injury had higher mortality than those without cardiac injury [] In this review we also summarized the possible relationship between basic heart disease and further cardiac injury [] Table In a cohort of COVID19 patients from Renmin Hospital of Wuhan University China Shi demonstrated that cardiac injury occurred in patients during hospitalization of which had basic heart disease including coronary heart disease and chronic heart failure And only patients with basic heart disease of COVID19 patients without cardiac injury [] Similarly Liu suggested that patients with basic heart disease in COVID19 patients had Table Comorbidity with cardiac injury in COVID19 patients with basic heart disease Subjects with COVID19 Proportion of basic heart disease Patients with Cardiacinjury Study Shi [] Liu [] Xu [] Ma [] Guo [] Patients with basic heart disease With cardiac injury Without cardiac injury cardiac injury compared with patients with basic cardiovascular diseases of COVID19 patients without cardiac injury [] Other studies also indicated that the patients with basic cardiovascular disease are more likely to present heat injury in COVID19 patients [ ] In view of the points above COVID19 patients with underlying cardiac conditions seem to have higher rates of cardiac injury SARSCoV2 infection and kidney injury Recently autopsy analysis on six COVID19 patients showed that varying degrees of acute tubular necrosis were observed in all the renal specimens Nucleoprotein NP antigens and NP positive inclusion body of SARSCoV2 could be seen in kidney tissues from all the samples Moreover viruslike ps were seen in kidney tissues by transmission electronic microscope TEM [] Su analyzed kidney abnormalities in autopsies of patients with COVID19 and found that diffuse proximal tubular damage with the loss of brush border were BiomedicinePharmacotherapy13120201106784 0c SARSCoV2 infection and liver injury M Dong observed Further investigation showed that diffuse necrosis can be seen under the light microscope and electron microscopic examination also showed the clusters of coronavirus ps with distinctive spikes in the tubular epithelium and podocytes [] It was reported that both NP antigens and RNA of SARSCoV2 were detected in urine of COVID19 patients [] These data coincide with the finding of the SARSCoV2 invasion in kidney Collectively SARSCoV2 could directly infect human renal tubules and lead to kidney damage Recent studies have shown that the incidence of acute kidney injury AKI in COVID19 patients ranged from and higher frequency of renal function damage with elevated blood urea nitrogen BUN or serum creatinine Scr was observed in COVID19 patients [ ] Table A study of patients with COVID19 indicated that levels of BUN and Scr were increased in and patients with COVID19 respectively And routine urine tests were performed on patients among which patients were positive for urinary protein and patients were positive for hematuria [] Another study also showed that about patients with COVID19 had abnormal renal function [] Moreover COVID19 patients with more severe disease progression have higher rates of AKI Huang and colleagues reported that of patients with AKI in the ICU were observed and none of the patients who did not require care in the ICU suffered AKI [] Xu found that the fatality rate was obviously higher in COVID19 patients with AKI than those without renal injury [] Furthermore in another study investigating patients with COVID19 at hospital admission more severe patients had higher rates of AKI and the Cox regression analysis also suggested that COVID19 patients who developed AKI had a significantly higher mortality risk [] Therefore AKI is more prevalent in severe cases with COVID19 An autopsy report of a 50yearold patient with COVID19 showed moderate microvesicular steatosis and mild lobular activity in liver tissues [] Moreover Zhao used human liver ductal anoids as a tool to investigate the SARSCoV2 infection and the tissue damage induced by SARSCoV2 ex vivo and the results showed that the expression of SARSCoV2 NP was easily detected in the patchy areas of the hepatic duct indicating that liver ductal anoids were susceptible to SARSCoV2 infection [] In addition SARSCoV2 infection could disrupt the barrier and bile acid transporting functions of cholangiocytes which indicated that SARSCoV2 might directly induce cholangiocyte injury and consequently bile acid accumulation [] In view of the points above liver damage in the COVID19 patients might be directly caused by the viral infection Abnormal liver functions were frequently reported in COVID19 patients [] Epidemiologic studies showed that almost half of the patients had differing degrees of liver damage [ ] Table Chen reported that out of patients had elevated alanine aminotransferase ALT patients had elevated aspartate aminotransferase AST and had elevated total bilirubin TBIL in Wuhan Jinyintan Hospital Wuhan China [] Similarly a nationwide study involving patients with COVID19 in China showed that more than of patients had elevated ALT and AST and of patients had elevated TBIL [] It was revealed that the levels of direct bilirubin indirect bilirubin ALT alkaline phosphatase ALP and gammaglutamyltransferase GGT were significantly higher in males than that in females with COVID19 and multivariate logistic regression analysis showed that male was an important independent risk factor for predicting acute liver injury ALI in COVID19 patients [] These data indicated that male patients with COVID19 may be more susceptible to liver injury Furthermore Table Characteristics of acute kidney injury after SARScid0 CoV2 infection Study Chen Wang Huang Guan Xu Preexisting kidney conditions NA NA NA [] [] [] [] [] Country China China China China China Subject Li [] Chen [] Hong [] Cheng [] Xiao [] Richardson [] Wan Li Qian Pei [] [] [] [] China China Korea China China America China China China China NA NA NA NA NA NA Scr Serum creatinine BUN Blood urea nitrogen AKI Acute kidney injury Abnormal renal functional indices Scr BUN NA Scr Scr Scr Scr BUN NA Scr BUN Scr NA NA NA Scr BUN Scr NA AKI NA Clinical classification of AKI NA NonICU cases ICU cases ICU cases Nonsevere cases Severe cases Mild cases Severe cases Critical ill cases Nonsevere cases Severe cases Recovered cases Died cases NonICU cases ICU cases NA Nonsevere cases Severe cases Cured cases In hospital cases Died cases Mild cases Severe cases Nonsevere cases Severe cases NA Moderate cases Severe cases Critically ill cases BiomedicinePharmacotherapy13120201106785 0cM Dong Table Characteristics of liver injury after SARScid0 CoV2 infection Study Country Subject China China China China China China China Korea America China China Chen [] Wang Huang Guan [] [] [] Xu Li [] [] Chen Hong [] [] Richardson et Wan Li al [] [] [] Qian [] China NA Patients with preexisting liverconditions NA NA NA Patients with abnormal liver functional indices ALT AST TBIL NA AST AST ALT TBIL ALT AST ALT AST TBIL ALT AST ALT AST TBIL AST ALT AST ALT AST TBIL ALT AST Abnormal liver functional indices in the Nonsevere patients AST ALT NA NA TBIL NA Abnormal liver functional indices in the severe patients ALT NA TBIL NA AST NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Nonsevere patients include patients without ICU care and recovered patients Severe patients include patients with ICU care and death ALT alanine aminotransferase AST aspartate aminotransferase TBIL total bilirubin multiple studies found that AST ALT and TBIL were significantly higher in patients treated in the ICU than that in nonICU patients [] Li suggested that among the patients with abnormal liver function moderate and severe types of patients were more likely to have liver injury and respectively [] Fu analyzed the relationship between ALI and mortality risk in COVID19 patients and the results showed that ALI is more common in the critically ill patients and ALI at the early stage increased death risk of COVID19 patients [] Together abnormal liver functions might be associated with the severity of patients with COVID19 SARSCoV2 infection and digestive tract injury Epithelial cells of the esophagus stomach duodenum and rectum in one COVID19 patient tested positive for SARSCoV2 RNA and the staining of viral NP was also visualized in the cytoplasm of epithelial cells in stomach duodenum and rectum [] Moreover minimally invasive autopsies were performed on three patients died of COVID19 and the results showed that some epithelial cells of the gastrointestinal mucosa were degenerated necrotic and detached [] These studies strongly supported that SARSCoV2 may directly infect the epithelial cells of digestive tract Table SARScid0 CoV2 detection in gastrointestinal specimens Study Subject [] [] Xiao Zhang Tan [] Xing Young Holshue Lescure Tang Wang Xu [] [] [] [] [] [] [] Gastrointestinal samples Stool Anal swabs Rectal swab Stool Stool Stool Stool Stool Stool Rectal swabs Tested positive in gastrointestinal specimens The positive time in gastrointestinal specimens days NA 6cid0 1cid0 5cid0 NA 3cid0 Positive time for Gastrointestinal samples after respiratory samples were negative days NA NA 8cid0 NA NA NA NA NA 2cid0 BiomedicinePharmacotherapy13120201106786 0cM Dong Multiple studies have identified that the SARSCoV2 RNA was detected in anal swabs [] rectal swabs [] and stool specimens [ ] of COVID19 patients It has been demonstrated that SARSCoV2 RNA could be detected in feces from more than half of COVID19 patients [] In another study Xing reported that SARSCoV2 RNA was detected in the feces of three pediatric cases with COVID19 in Qingdao China and the persistence of SARSCoV2 in the digestive tract lasted for 6cid0 days [] The possibility of fecaloral transmission of SARSCoV2 infection needs to be taken into account Furthermore as shown in Table long duration of SARSCoV2 detection in digestive tract by RTPCR has been reported and viral RNA remained detectable in the digestive tract for 2cid0 days after nucleic acid turned negative in respiratory samples [] The studies suggested that SARSCoV2 could be detected from respiratory tract specimens during the early period to digestive tract specimens during the late period and viral nucleic acid tests in both the respiratory and digestive tract are necessary to confirm the complete clearance of virus Some COVID19 patients presented gastrointestinal symptoms such as diarrhea nausea vomiting and abdominal pain [] Holshue reported the first case of COVID19 patient in the USA which had nausea and vomiting before admission [] Multiple studies found that gastrointestinal symptoms including diarrhea nausea and vomiting and abdominal pain were common at presentation in COVID19 patients [ ] Table In a cohort of patients with COVID19 in Wuhan China gastrointestinal symptoms were described in up to [] Moreover Sun showed that critically ill patients with COVID19 had gastrointestinal injury GI during hospital stay and the survival curves showed that the mortalities of patients with GI was greater than that of patients without GI [] Jin also found that the rate of the severe type was markedly higher in COVID19 patients with GI symptoms than that in those without GI symptoms [] These data suggested that GI is one of the common extrapulmonary an injuries in COVID19 patients and may be related to the severity of the disease On the other hand many studies showed that patients with COVID19 could present initially with the typical gastrointestinal sympto | 2 |
Identification and treatment of obstructive sleep apnea by a primarycare team with a subset focus on chronic painmanagement within the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existBackgroundPatients diagnosed with obstructive sleep apnea OSA who also consume prescription opioids have a greater likelihood of morbidity and mortality This study evaluated whether a primary care team focused on chronic pain care management could use a validatedquestionnaire STOPBang and motivational followup to increase identification and treatment of OSAMethodsThis study was a retrospective dual arm prepost controlled study Participants of thisstudy included the complete chronic pain management sub group treated by this primarycare team Participants were � years old and prescribed daily opioids for treatment ofchronic pain All participants had a multifaceted individualized educational meeting thatincluded completing a STOPBang questionnaire Participants who received a score �three were advised to follow up with their primary care physician Participants were seenquarterly throughout the studyResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of months 18month average postintervention vs CPAPuse in the control group months of observation both groupswere chronic opioid users with OSA This was a relative improvement p Asecondary outcome was that of nonprior CPAP users obtained CPAP post intervention a prepost improvement p x2 with degree of freedom Alsoparticipants who were likely to have OSA STOPBang score � or had a positive polysomnography AHI with comorbidities compared to those unlikely to have OSA STOPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamBang score or had a negative polysomnography AHI in this study were more likelyto be male have a higher BMI have hypertension have cardiovascular disease andorhave diabetes all typesConclusionTeam based care management for participants taking prescription opioids where STOPBang questionnaires were completed were associated with an increase in the identificationand treatment of OSAIntroductionStudy rationaleThe objective of this study was to evaluate whether a teambased care management intervention would increase identification and treatment of obstructive sleep apnea in participants taking medication for chronic painCurrent knowledgeObstructive sleep apnea OSA is a chronic sleep disorder characterized by episodes of apneasand hypopneas or the complete or partial collapse of the upper airway [] A diagnosis ismade when a patient has or more of these events in an hour or five of these events if otherpredicting symptoms for OSA are present [] Approximately million American adults havethis sleep disorder Patients with moderate to severe OSA are often treated with a continuouspositive airway pressure device or CPAP Treatment can reduce the risks of hypertension coronary artery disease heart failure arrhythmias sudden cardiac death and stroke [] A validated questionnaire STOPBang score can be used as a screening tool to identify patients atrisk for OSA A STOPBang sleep questionnaire is commonly distributed to patients whoshow signs of having potential risk factors for OSA STOP questions snoring tirednessobserved apnea and high blood pressure and Bang BMI kgm2 Age years Neckcircumference cm and Gender male [ ] A score of three or higher on STOPBangplaces patients at higher risk of OSA requiring further evaluation which includes an overnightpolysomnogram or sleep study to confirm diagnosis []Prescription opioid use for pain management is still a common practice in primary care settings though the rates of overdose due to these drugs in the United States are at an unprecedented high with deaths in [] Patients using opiates for chronic painmanagement have also been found to have a greater likelihood of developing central sleepapnea which worsens the severity of OSA and ataxic breathing [] A relationship existsbetween pain and sleep disruption with over of patients who have chronic pain havingreported difficulty sleeping which increases hyperalgesia [ ] An analgesic effect may bepresent where pain creates sleep disorders and a shortage of sleep increases pain [ ]Patients with OSA who are then prescribed opioids have an increased risk of opioidinducedrespiratory depression OIRD [] Despite the potential morbidity of the combination ofOSA central sleep apnea and Opioid use studies have shown that those that have this combination only remain on definitive treatment for their OSA CPAP of the time [] It istherefore of the utmost importance that patients who chronically use opioids are evaluated forOSA and those diagnosed adhere to CPAPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIn order to decrease mortality it is necessary to educate patients on the risks and healthconcerns related to taking opioids Brief interventions can influence behavior of patients whoare at high risk for abuse of a substance [] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] These individualized information gathering motivational meetings allow patients to experience empathy as their medical needs are met [ ] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] Instructionand guidance can be coupled with an assessment of a patients potential risk factors for comorbidities such as OSA using a STOPBang questionnaireMaterials methodsDesignThis study was a retrospective dual arm prepost controlled study to evaluate whether anintervention STOPBang questionnaire education and motivational followup wouldimprove OSA diagnosis and treatment compliance The first investigation arm compared thecontrol group daily opioid use with OSA to the part of the intervention group with OSA onCPAP or at risk for OSA STOPBang � This arms purpose was to determine whether theintervention increased treatment compliance with CPAP The second investigation armcompared the study group to itself prepost intervention This arm evaluated whether applyingthe intervention increased diagnosis and treatment of OSA The single independent variablein this study was a participants admission to the chronic pain management subgroup withthe administration of a STOP Bang questionnaire and the dependent variable was CPAP useInclusion criteria consisted of ongoing participation in primary care pain management subgroup daily opioid use and an age � This study occurred between October and January which included monthsfor exposure and then at least months of follow up One of Intermountain Healthcares outpatient facilities in Utah served as the site for this study The total number of patients duringthe duration of the study who entered into the pain management sub group � years oldand were taking a daily dose of opioids was Out of the identified participants participants remained in the clinic during the entirety of this study Fig Ethics approvalIRB Number for this study was obtained from the Institutional Review Board ofIntermountain Healthcare Consent was waived for this retrospective studyControlsA Medline and Pubmed basic search from was used to identify a historical yet contemporary United States based control group that had Obstructive Sleep Apnea and met theinclusion criteria of being � years old and were taking a daily dose of opioids The controlthat was chosen was comparable to this studys intervention group with regard to Race AgeGender Morphine Equivalent Daily Dose and Comorbidities Also the control used camefrom a study that lasted a similar duration []SubjectsEach participant received an information gathering and educational session with a registerednurse in the primary care facility at the time of their admittance into the chronic pain management program between During this session a full pain history was obtained fromparticipants All participants in the study underwent STOPBang screening All participantswho were at high risk for OSA STOPBang � were referred back to their primary carePLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamFig Participant retention101371journalpone0237359g001physician Participants and physicians made joint decisions during a followup appointmentwhether to pursue polysomnography Reasons why participants did not complete polysomnography or attain treatment for OSA are that the participants refused workup there was ashared decision due to participant frailty had cancer end stage or were noncompliant toCPAP Fig Fig participants at high risk for sleep apnea STOPBang score � who did not undergo diagnostic evaluation orcomplete treatment101371journalpone0237359g002PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamEach participant received initial inperson education with the same registered nurse regarding nonpharmacological treatment of pain selfhelp groups risks and benefits of treatment ofpain management compliance with treatment of pain and its comorbidities and informationabout Naloxone an opioid reversal agent This education was provided in a hour sessionusing the FRAMES Feedback Responsibility Advise Menu for Change Empathy andEnhancing Self Efficacy motivational method [] A chronic pain management agreementwas executed Additional information obtained during this session included a Current OpioidMisuse Measure and a STOPBang questionnaire All participants who received a score greaterthan or equal to three on the STOPBang were told to follow up with their primary care physician All primary care providers were made aware they would receive electronic data onSTOPBang questionnaires and participants with scores � would make a follow up appointment with them Upon completion of this session information was recorded electronically andtransmitted to the primary care physician on record Each participant also had quarterly inperson followup appointments with their primary care physician In addition quarterly inperson or phone visits with the pain management nurse were continued Physician visits varied in length and scope Individualized nursing visits that continued to use the FRAMESmethod and lasted for a duration of minutes were focused on remaining compliant with allongoing therapies The nurse would document the interaction and any concerns about compliance with any therapy in the patients EMR Electronic Medical Record at the conclusionof the visits The information was based on the subjective input received from the participantQuarterly followup remained consistent throughout the study Attending all followupappointments both with the primary care physician and the pain management nurse was aprerequisite to remain in the pain management group Remaining in the pain managementwas group was the only avenue to receive controlled pain medication at this facilityData collectionThe baseline biometric parameters of Race Gender BMI Age Tobacco Use Alcohol Use andMorphine Equivalence Daily Dosage MEDD were extracted from each participants electronic medical record EMR Collected data also included information on Chronic DiseaseHypertension Cardiovascular Disease Pulmonary Disease Thyroid Disease Diabetes Gastroesophageal Reflux Disease and Chronic Kidney Disease and specific medication typesBenzodiazepines and Pregabalin STOPBang scores were gathered during the initial chronicpain management encounter when participants joined the study Subjective data on CPAPadherence was found in the EMR documentation of the pain management nurse and primarycare physician follow up reports postinclusion in the studyStatistical methodsMeans and standard deviations were calculated for demographic variables The DAgostinoPearson test was used to confirm normality [] Unequal variance ttesting was used in the statistical analysis of both arms of this study to compare Age BMI and Morphine EquivalentDaily Dosing Two proportion Ztesting was used to compare the control group daily opioiduse with OSA to the participants in the intervention group with OSA on CPAP or at highrisk for OSA STOPBang � with regards to CPAP use Race Gender Diabetes Cardiovascular Disease and Hypertension Two proportion Ztesting was also used to compare the participants not likely to have OSA STOPBang or had a negative polysomnography to thosewith OSA on CPAP or at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP A x contingency table using McNemars test with correction for continuity was used to compare the participants CPAP use prepost intervention []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIRB approvalIRB Approval was granted from Intermountain Healthcare IRB IRB Number ResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of months 18month average postintervention vs CPAP use in the control group months of observation which were chronic opioid users with OSA This was a relative improvement p A secondary outcomewas that of nonprior CPAP users obtained CPAP post intervention a prepostimprovement p x2 with degree of freedom Fig This resulted in ofintervention participants being treated with CPAP Comparing the average number of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the control dataset resulted in significant differences for BMI and Male Gender Otherdemographics between these two groups Age p MEDD p Race Caucasianp Hypertension p Cardiovascular Disease p and Diabetes all formsp showed no statistically significant differences between groups Comparing the averagenumber of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the average number of intervention participants not likely to have OSA STOPBang orhad a negative polysomnography resulted in the following statistically significant differencesBMI p Male Gender p00001 Hypertension p Cardiovascular Diseasep and Diabetes all forms p Other demographics between these two groupsAge p MEDD p Race Caucasian p Benzodiazepine Use p Pregabalin Use p and Active Chronic Disease States Gastroesophageal Reflux p Chronic Kidney Disease Thyroid Disease p and Pulmonary Diseasep showed no statistically significant differences between groups Table DiscussionMain findingsCurrent high death rates compared to healthy subjects of OSA and chronic pain managementwith daily opioid use warrant efforts to improve care [] This studys findings can be anFig CPAP use before and after intervention101371journalpone0237359g003PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamTable Comparison of clinical characteristics S1 AppendixDemographicsAverage Number of Participants withOSA on CPAP or at risk for OSASTOPBang � Control DailyOpioid Use withOSAAgeBMI ���Morphine Equivalent mgdat Conclusion of Study � months Pvalue Average Number of Participants Not Likelyto Have OSA STOPBang or NegPolysomnography PrePostPvalueDemographicsAverage Percentage of Participantswith OSA on CPAP or at risk for OSAControl DailyOpioid Use withPvalueAverage Percentage of Participants NotLikely to Have OSA STOPBang orPrePostPValueSTOPBang � OSANeg PolysomnographyRace CaucasianGender Male��Diabetes���Cardiovascular Disease��Hypertension�Gastroesophageal Reflux DiseaseThyroid Disease All FormsPulmonary Disease All Varieties Active CancerChronic Kidney DiseaseConcurrent Benzodiazepine UseConcurrent Pregabalin Use� p value of �� p value of ��� p value of 101371journalpone0237359t001NANANANANANANA NANANANANANANAimportant step towards improving mortality and morbidity when these two diseases are combined The results suggested that using a STOPBang questionnaire accompanied with motivational education initially as well as in followup improved diagnosis and treatment of OSAThese findings suggest that teambased care can positively impact the outcome of patientswith OSA when they are concurrently on chronic daily opioids and CPAP These findings warrant additional research Diagnosed sleep apnea participants who were using CPAP had a significantly higher rate of being male having hypertension having cardiovascular diseasehaving diabetes all types and having a higher BMI The fact that of participants at riskfor sleep apnea STOPBang � did not receive a full evaluation or end up on definitive treatment highlights the need for continued effort in this fieldLimitationsThe data found in this study regarding CPAP adherence was subject to limitations as wellSome participants that joined this teambased care management approach to treat chronicpain had already been prescribed CPAP and their adherence to the treatment was included inthis studies data set possibly overestimating the conclusions Adherence to CPAP in this studywas based on subjective treatment reconciliation at followup appointments with physiciansand nurses and since many participants received their sleep treatment from outside sourcesCPAP adherence data other than confirmation from the patient was not available If a participant indicated that they had not been using their CPAP � of the time between anyPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamfollowup appointments after the initiation of treatment they were considered to be nonadherent This type of data collection is subject to significant recall bias and could potentiallyinflate the percentage of participant adherenceThis preliminary research was done in the format of a prepost dual arm retrospectivestudy This type of study has inherent and significant limitations First due to the nature of theintervention team members nurses and physicians were not blinded and continued to evaluate participants throughout the course of this study and could have recommended evaluationand therapy of sleep apnea for alternative reasons This study could have been affected by aherd mentality since the primary care physicians worked together in one group and theircomposite style could have increased or decreased the identification and treatment of sleepapnea These limitations could be mitigated by performing a large prospective multicenterstudyThis study is also limited due to the nature of using a historical control [] Although the control used mimicked the experimental group in most ways daily opioid use MEDD race age andpercentages of diabetes hypertension and cardiovascular disease it varied in both BMI controlgroup had a higher average BMI and gender control group had a greater number of male participants It has been previously demonstrated that a higher BMI favors adherence to CPAP [] This does not detract from the finding that the intervention group in this study which had alower BMI had a higher adherence to CPAP Gender has also been evaluated in previous CPAPcompliance studies Two studies have shown no difference between genders whereas a differentstudy showed an increase in male noncompliance and another where there was an increase inmale compliance [] As there is no definitive determination from these studies it is possiblethat the variance between the intervention and control groups were due to the gender differenceIt would seem from the above studies that there was no strong trend resulting from gender making gender unlikely to be the cause of the preponderance of the varianceThere was a loss of followup with primary care physicians after the administration of theSTOPBang questionnaire in this study Fig Patients may have fotten to or chosen notto schedule a followup appointment with their primary care physician after being determinedhigh risk for sleep apnea The participant received education on six other aspects of opioidmanaged pain during the one hour informational meeting Another limitation was that thedata from the intervention session was transmitted enblock to the primary care physicianelectronically These limitations could have been improved by having the nurse set up a specific appointment to consider sleep disorders in the highrisk group and to either verbally orelectronically deliver this information in an isolated approachComparisonsThis study reaffirmed previous studies that showed STOPBang was a tool that could identifyOSA [ ] Other similarities to previous investigations were found In those studies wheredaily opioid dosing for pain management was present of patients were found to sufferfrom OSA and of patients were found to be compliant to CPAP [ ] The data rangefor previous studies in regard to OSA incidence is consistent with this study in which ofparticipants were either diagnosed with OSA on CPAP or were at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP This study was significantly higher with regards to the percentage of remaining on CPAP suggesting thatdiligence in repetitive diagnosis attempts and continued follow with motivational techniquesmay improve care in this type of patient Also this study like others suggested p thathypertension cardiovascular disease diabetes male gender and higher BMI are more common in patients with OSA []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamConclusionsLeft untreated sleep apnea has a high rate of morbidity and mortality The use of prescribedopioids as a form of chronic pain management in a primary care setting can increase apatients likelihood of having sleep apnea A teambased intervention that included theadministration of a STOPBang questionnaire was associated with an increased diagnosis ofOSA prepost improvement level It was also associated with an increased adherenceto CPAP relative improvement The primary reasons CPAP treatment was not receivedincluded refusal of evaluation nonadherence to CPAP or shared physicianpatient decisionbased on agefrailtySupporting informationS1 Appendix Raw study data setXLSXAuthor ContributionsConceptualization Kathleen Whittington Leigh Simpson Dixie HarrisData curation Kathleen Whittington Michael ClayFormal analysis Kathleen Whittington Michael Clay Dixie HarrisInvestigation Joanna TierneyProject administration Joanna TierneySupervision Dixie HarrisWriting original draft Kathleen WhittingtonWriting review editing Kathleen Whittington Leigh Simpson Michael Clay JoannaTierney Dixie HarrisReferencesFarney Robert J The STOPBang equivalent model and prediction of severity of obstructivesleep apnea relation to polysomnographic measurements of the apneahypopnea index Journal ofclinical sleep medicine JCSM official publication of the American Academy of Sleep Medicine vol 65B 105664JCSM1306 PMID Manne Mahesh B Obstructive Sleep Apnea Who Should Be Tested and How ClevelandClinic Journal of Medicine Aug wwwmdedgecomccjmarticle105363cardiologyobstructivesleepapneawhoshouldbetestedandhow Abrishami A Khajehdehi A Chung F A systematic review of screening questionnaires for obstructivesleep apnea Can J Anaesth 101007s126300109280x PMID Vasu TS Doghramji K Cavallazzi R Obstructive sleep apnea syndrome and postoperative complications clinical use of the STOPBANG questionnaire Arch Otolaryngol Head Neck Surg 101001archoto20101020 PMID Kumar S Mcelligott D Goyal A Baugh M Ionita RN Risk of Obstructive Sleep Apnea OSA in Hospitalized Patients Chest 1384779A Ong TH Raudha S FookChong S Lew N Hsu AA Aal H Simplifying STOPBANG use of a simplequestionnaire to screen for OSA in an Asian population Sleep Breath Drug Overdose Deaths Centers for Disease Control and Prevention Centers for Disease Control andPrevention June wwwcdcgovdrugoverdosedatastatedeathshtml Substance Abuse and Mental Health Services Administration Drug Abuse Warning Network National Estimates of DrugRelated Emergency Department Visits HHS Publication No SMA PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team DAWN Series D39 Rockville MD Substance Abuse and Mental Health Services Administration Schneiderhan JMD Clauw DMD Schwenk TMD Primary care of patients with chronic pain J Am MedAssoc Walker James M Chronic Opioid Use Is a Risk Factor for the Development of Central SleepApnea and Ataxic Breathing Journal of Clinical Sleep Medicine JCSM Official Publication of theAmerican Academy of Sleep Medicine American Academy of Sleep Medicine Aug wwwncbinlmnihgovpmcarticlesPMC1978331 PMID Webster LR Choi Y Desai H Webster L Grant BJ Sleepdisordered breathing and chronic opioid therapy Pain Med 101111j15264637200700343x PMID Onen SH Onen F Courpron P Dubray C How pain and analgesics disturb sleep Clin J Pain 10109701ajp000012975731856f7 PMID Cozowicz Crispiana Opioids for Acute Pain Management in Patients with Obstructive SleepApnea Anesthesia Analgesia vol no pp 101213aneJaoude Philippe Lal Ashima Vermont Leaj Porhomayon Jahan Ali A ElSolh Pain Intensity and Opioid Utilization in Response to CPAP Therapy in Veterans with Obstructive Sleep Apnea on Chronic Opioid Treatment Journal of Clinical Sleep Medicine Barbor TF HigginsBiddle JC World Health anization Dept of Mental Health and SubstanceDependence Brief 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Marshall NS Wong KK Sleep apnea as an independent risk factor for allcause mortality the Busselton Health Study Sleep Aug PMID Liang Y Turner B Assesing Risk for Drug Overdose in a National Cohort Role for Both Daily and TotalOpioid Dose J Pain Apr 101016jjpain201411007 PMID Heejin Kim MinSukim Treatment Outcomes and Compliance According to Obesity in Patientswith OSA European Archives of OtoRhinoLaryngology PelletierFleury N Rakotonanahary D Fleury B The age and other factors in the evaluation of compliance with nasal continuous positive airway pressure for obstructive sleep apnea syndrome A Coxsproportional hazard analysis Sleep Med 101016s1389945700 PMID Anttalainen U Saaresranta T Kalleinen N Aittokllio J Vahlbergy T Polo O CPAP adherence and partial upper airway obstruction during sleep Sleep Breath 101007s1132500701025 PMID Sin DD Mayers I Man GC Pawluk L Longterm compliance rates to continuous positive airway pressure in obstructive sleep apnea a populationbased study Chest 101378chest1212430 PMID Ye Lichun Pien Grace W Ratcliffe Sara J Weaver Terri E Gender Differences in Obstructive SleepApnea and Treatment Response to Continuous Positive Airway Pressure J Clin Sleep Med PMID PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team Mador M Henderson J Effect of Opioids on Sleep and Breathing in Chronic Pain Patients Journal ofClinical Sleep Medicine Aug 105664jcsm3952 PMIDPLOS ONE 101371journalpone0237359 August PLOS ONE 0c' | 2 |
"Vitamin D is a fatsoluble vitamin vitamin D is essential to sustain health and it protects againstosteoporosis It is crucial to the human bodys physiology in terms of muscular movement and neurological signaltransmission and to the immune system in defense against invading pathogensCase presentation This was a case of a 26yearold Sudanese woman who presented with a 2year history ofanosmia recurrent nasal polyps back pain and chronic fatigue She was diagnosed as having a case of vitamin Ddeficiency and responded well to treatmentConclusion There is an association between vitamin D deficiency and recurrent allergic nasal conditionsKeywords Vitamin D deficiency Allergy Nasal polyps Backache Chronic fatigabilityBackgroundVitamin D is a fatsoluble vitamin it is naturally presentin some foods and as dietary supplements It is also produced endogenously through exposure to ultraviolet raysfrom sunlight Vitamin D obtained from sun exposurefood and supplements is biologically inert and mustundergo two hydroxylations in the body for activationTheand produces hydroxyvitamin D 25OHD also known as calcidiolThe second occurs in the kidney and forms the physiologically active 125dihydroxy vitamin D 125OH2D alsoknown as calcitriol []first occursin theliverVitamin D is found in cells throughout the body vitamin D is essential to sustain health and it protectsagainst osteoporosis It is crucial to the human bodysphysiology in terms of muscular movement and neurological signal transmission and to the immune system indefense against invading pathogens []Although there are different methods and criteria fordefining vitamin D levels the criteria Holick proposed Correspondence drmuhanadkamalhotmailcom1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum SudanFull list of author information is available at the end of the have been widely accepted In this proposal vitamin Ddeficiency is defined as blood level of less than ngmlinsufficiency of vitamin D is defined as blood levels ranging between and ngml and sufficiency if greaterthan or equal to ngml [] About one billion peopleglobally have vitamin D deficiency and of the population has vitamin D insufficiency The majority ofaffected people with vitamin D deficiency are the elderlyobese patients nursing home residents and hospitalizedpatients Vitamin D deficiency arises from multiplecauses including inadequate dietary intake and inadequate exposure to sunlight Certain malabsorption syndromes such as celiac disease short bowel syndromegastric bypass some medications and cystic fibrosis mayalso lead to vitamin D deficiency []Vitamin D deficiency is now more prevalent than everand should be screened in highrisk populations Manyconflicting studies now show an association betweenvitamin D deficiency and cancer cardiovascular diseasediabetes autoimmune diseases and neuropsychiatricdisorders [ ] The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of two functional endoscopic sinusCase presentationThis was a case of a 26yearold Sudanese woman married who has a 3yearold boy This woman presentedto our ear nose and throat ENT department complaining of anosmia for the past years She had a history ofsurgeriesFESSs for nasal polyps the first one was years agoand the second one was years prior to presentationShe complained of being highly sensitive to different irritants including dust weather change perfumes andpetsShe also stated that she attended more than three different physicians due to generalized fatigue and gettingtired easily after simple daily activity in addition to sleeping for more than hours a dayShe attended an orthopedic clinic for unspecified lower back pain that was notrelated to any type of trauma or physical activity a lumbosacral magnetic resonance imaging MRI was done andrevealed no abnormal findingsShe mentioned that she isknown to be anxious most of the time and aggressive toward simple reactions from her family members She hadno psychiatric history and was not using any medicationsShe was not known to be diabetic or hypertensive orto have any chronic illnesses she was not on any regularmedication She is a housewife of high socioeconomicstatus she is well educated graduated from dentalschool with a bachelors degree but currently notemployed She has never consumed tobacco or alcoholshe practiced regular cardio exercisesOn examinationshe looked healthy well not pale or jaundiced Herpulse rate was 74minute and her blood pressure was Her body mass index BMI was All systems examinations were normal except for bilateral nasalpolyps Complete blood count CBC renal function testREF electrolyte liver function test LFT thyroid function test TFT urine analysis general urine test antinuclear antibody ANA and rheumatoid factor RFwere all normal An imaging profile included lumbosacral MRI a computed tomography CT scan of her sinuses and electrocardiogram ECG which were normalexcept for bilateral nasal polyps and severe sinusitis thatlooked allergic to fungi in natureShe underwent FESSsurgery to remove the polyps and clean out her sinusesup to weeks after surgery she used nasal steroidsmometasone furoate two times a day but hersymptoms regarding anosmia were not improved MRIof her brain and a CT scan of her sinuses were done andboth revealed normal features A vitamin D deficiencywas suggested and the laboratory results revealed a lowvitamin D level of ngml Treatment with vitamin Dsupplement was prescribed at international unitsIU weekly for weeks and then IU maintenancedose daily she was advised to take food rich in vitaminD and get exposed to sunlight for minutes three timesa week after the loading dose of supplement She was atregular followup for months at rates of weekly for thefirst month every weeks for the second month andmonthly for the rest of the followup period At eachvisit she was assessed with clinical history and examination It was noticed that the symptoms of tirednesssleeping anosmia and back pain were dramatically improving during that period At the months followupher blood level of vitamin D was normal she describedher condition as free from all symptoms and shereturned back to normal physical activityDiscussion and sThis was a nonclassical case of vitamin D deficiency ofa 26yearold woman who presented with chronic anosmia and recurrent nasal polyps She was diagnosed ashaving a case of vitamin D deficiency and respondedwell to vitamin D replacement therapy This case correlated an association between decreased levels of vitaminD and recurrent nasal polyps that led in time to chronicanosmia as a result of chronic high sensitivity reactionstriggered by our patients autoimmune system The literature links chronic rhinosinusitis with nasal polypsCRSwNP with asthma and allergic rhinitis but the cellular and molecular mechanisms that contribute to theclinical symptoms are not fully understood Sinonasalepithelial cell barrier defectsincreased exposure topathogenic and colonized bacteria and dysregulation ofthe host immune system are all thought to play prominent roles in disease pathogenesis []Despite all the previous surgical and medical interventions over the past years our patients condition did notimprove and she still complained of anosmia A study revealed that this patient was experiencing excessive allergicreactions that led to recurrent nasal polyps It is wellknown that classical clinical effects of vitamin D deficiencyare bones and musculoskeletalrelated disorders severallines of evidence demonstrate the effects of vitamin D onproinflammatory cytokines regulatory T cells and immune responses with a conflicting interpretation of theeffects of vitamin D on allergic diseases []The working diagnosis was suggested in relation tosome musculoskeletal symptoms and chronic fatigue especially when the imaging profile for her lower back andall routine investigations were normal It has been suggested that clinicians should routinely test for hypovitaminosis D in patients with musculoskeletal symptomssuch as bone pain myalgias and generalized weaknesswhich might be misdiagnosed asfibromyalgia andchronic fatigue [] The most common causes of anosmia were assessed as well and they were negative theseincluded sinonasal diseases post infectious disorder andposttraumatic disorder and congenital defects and disorders caused by neurodegenerative disease [] 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of Authors contributionsMI analyzed and interpreted the findings of the case report and was themajor contributor in writing the manuscript ME reviewed the report andadded valuable comments All authors read and approved the finalmanuscriptFundingNoneAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from Albasar Institutional Review BoardConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum Sudan 2Preventive Medicine and EpidemiologyAlneelain University Khartoum SudanReceived March Accepted July Thus blood level for vitamin D was requested and theresults were of low D levelIn the past history of the previous nasal polyps surgeries our patient noted that there was no anosmia and hermain complaints were classic complaints of sinusitis including sneezing nasal blockage and headache Soonafter surgery her symptoms improved except for theallergyrelated symptoms despite usage of inhaled steroids spray She stated that at the last time the presentation was different since it was only anosmia indicatingthat there was significant inflammation that affected thesmell receptors around the olfactory epithelium Afterthe last nasal polyps and sinuses drainage surgery thesymptoms related to allergic reactions including chronicsneezing did not improve for up to weeks and she wasstill suffering from hyposmia although that was a fairpostoperative period for recoveryThe symptoms of anosmia and sneezing and othersystematic symptoms gradually started to improve aftervitamin D supplements indicating that the main reasonbehind her symptoms was vitamin D deficiency She wasfollowed up for up to months after establishment ofvitamin D supplements and at the last followup she hada normal sense of smell and she was free from backpain fatigue and allergyrelated symptomsThis was a nonclassical presentation as our patientwas young and she did not have alkaline phosphatasecalcium and phosphorus abnormalities [] that are expected in cases of vitamin D deficiencyThis case revealed an association between decreasedlevels of vitamin D and recurrent nasal polyps that ledto anosmia as a result of hypersensitive reactions produced by the bodys systemsAlthough vitamin D deficiency is prevalent measurement of serum 25OHD level is expensive and universalscreening is not supported However vitamin D testingmay benefit those at risk for severe deficiencyIt is highly recommended to consider vitamin D deficiency among all patients with unspecified symptoms orin cases of nondiagnosed disorder regardless of the presenting complaintIn there is an association between vitaminD deficiency and recurrent allergic nasal conditionsAbbreviationsCRSwNP Chronic rhinosinusitis with nasal polyps CT Computedtomography ECG Electrocardiogram ENT Ear nose and throatFESS Functional endoscopic sinus surgery BMI Body mass indexCBC Complete blood count RFT Renal function test LFT Liver function testTFT Thyroid function test ANA Antinuclear antibody RF Rheumatoid factorIU International unitReferencesNational Institutes of Health Vitamin D fact sheet for health professionals httpsodsodnihgovfactsheetsVitaminDHealthProfessionalen2Accessed Apr National Institutes for Health NIH Vitamin D fact sheet for consumers httpsodsodnihgovfactsheetsVitaminDConsumer Accessed Dec Kuriacose R Olive KE Vitamin D insufficiencydeficiency managementSouth Med J httpsdoi101097SMJSizar O Khare S Givler A Vitamin D deficiency Treasure Island Stat PearlsPublishing httpswwwncbinlmnihgovbooksNBK532266 PMID Accessed Dec Wlliam B Fatme A Meis M Targeted 25hydroxyvitamin D concentrationmeasurements and vitamin D3 supplementation can have important patientand public health benefits Eur J Clin Nutr httpsdoi101038s4143002005640Hanmin W Weiwen C Dongqing L Xiaoe Y Xiaode Z Nancy O et alVitamin D and chronic diseases Aging Dis httpsdoi1014336AD20161021 Whitney W Ropert P Robert C Chronic rhinosinusitis with nasal polyps JAllergy Clin Immunol Pract httpsdoi101016jjaipThacher TD Clarke BL Vitamin D insufficiency Mayo Clin Proc httpsdoi104065mcp20100567Kennel KA Drake MT Hurley DL Vitamin D deficiency in adults when totest and how to treat Mayo Clin Proc httpsdoi104065mcp20100138Sanne B Elbrich M Duncan B Antje W Veronika S Joel D et al Anosmia aclinical review Chem Senses httpsdoi101093chemsebjx025Shikino K Ikusaka M Yamashita T Vitamin Ddeficient osteomalacia due toexcessive selfrestrictions for atopic dermatitis BMJ Case Rep httpsdoi101136bcr2014204558AcknowledgementsNot applicablePublishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 2 |
"researchWhat are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records dataHelen Strongman Rachael Williams2 Krishnan Bhaskaran1To cite Strongman a0H Williams a0R Bhaskaran a0K What are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records data BMJ 202010e037719 101136bmj 2020037719 º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received February Revised April Accepted July Authors or their employers Re use permitted under CC BY Published by BMJFor numbered affiliations see end of Correspondence toDr Helen Strongman helen strongman lshtm ac ukObjectives To describe the benefits and limitations of using individual and combinations of linked English electronic health data to identify incident cancersDesign and setting Our descriptive study uses linked English Clinical Practice Research Datalink primary care cancer registration hospitalisation and death registration dataParticipants and measures We implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and We calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers We described completeness of grade and stage information in the cancer registration data setResults gold standard cancers were identified Positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate Sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone For case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer When primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed Completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancersConclusions Ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority Strengths and limitations of this study º This is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in England to identify cases of the most common incident cancers º Using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º We described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º A key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataINTRODUCTIONThe Clinical Practice Research Datalink CPRD provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 Use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 In the field of cancer epidemiology CPRD primary care data linked to Hospital Episode Statistics Admitted Patient Care Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access data HES APC Office of National Statistics ONS mortality and National Cancer Registration and Analysis Service NCRAS cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment Use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for NCRAS data Research teams therefore commonly choose not to use all available linked data3 Cancer epidemiology studies can also be conducted using NCRAS and HES APC data provided by National Health Service NHS Digital and Public Health England PHE without linkage to CPRD primary care data4 This provides national coverage at the expense of the detailed health data that are available in primary care recordsValidation studies assessing concordance between CPRD GOLD HES APC and NCRAS data have estimated high positive predictive values PPVs for CPRD GOLD data and varying proportions of registered cancers that are not captured in CPRD GOLD and HES APC5 The most up to date analysis by Arhi et al included the five most common cancers and all papers focussed on concordance between CPRD GOLD only and NCRAS existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs National data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and Clinical Commissioning Groups10 We aim to describe and compare the benefits and limitations of using different combinations of linked CPRD primary care data HES APC ONS mortality and NCRAS cancer registration data for conducting cancer epidemiology studies Our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets We have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets We also describe the availability of stage grade and treatment variables over time in the cancer registration data for the CPRD linked cohort This reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesMETHODSStudy design and settingWe completed a concordance study using linked2 English CPRD GOLD HES APC ONS mortality and NCRAS data CPRD GOLD data were extracted from the January monthly release and the 13th update to CPRDs linked data The study period was from January to December with December matching the end of the NCRAS coverage periodThe CPRD GOLD database includes de identified records from participating general practices in the UK who use Vision software1 General practice staff can record cancer diagnoses using Read codes or in free text comments boxes though the latter are not collected by CPRD Diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care CPRD GOLD data are linked to HES APC ONS mortality and NCRAS through a trusted third party for English practices that have agreed to participate in the linkage programme2 HES APC data are collected by NHS Digital to co ordinate clinical care in England and calculate hospital payments12 Admissions for and related to cancer diagnoses are recorded using International Classification of Diseases version ICD10 codes National cancer registration data are collected by NCRAS which is part of PHE in accordance with the Cancer Outcomes and Services Data set13 which has been the national standard for reporting of cancer in England since January Data include ICD10 codes to identify the cancer site and more detailed information such as stage and grade ONS mortality data includes dates and causes of deaths registered in England recorded using ICD10 codesParticipants exposures and outcomesOur underlying study population included male and female patients registered in CPRD GOLD practices who were eligible for linkage to HES APC NCRAS and ONS mortality data and had at least days of follow up between January and December Start of follow up was defined as the latest of the current registration date within the practice and the CPRD estimated start of continuous data collection for the practice up to standard date End of follow up was determined as the date the patient left the practice ONS mortality date of death or practice last collection dateIdentification and classification of cancer codesWe used code lists to classify cancer records in each of CPRD GOLD HES APC and ONS mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https data Incompletely specified cancer codes could be mapped to cancer site eg ICD10 code C689 Malignant neoplasms of urinary an unspecified was considered consistent with both bladder and kidney cancer For NCRAS we accessed coded records for the most common cancers We included cancers recorded in the clinical or referral file for CPRD GOLD cancers recorded in any diagnosis field for HES APC and the underlying or Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Gold standard algorithm to identify incident site specific cancers using all data sources HES Hospital Episode Statistics NCRAS National Cancer Registration and Analysis Service ONS Office of National Statisticsmost immediate cancer cause of death in ONS mortality dataCancer case definitions based on individual sources and combinations of sourcesWe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources NCRAS alone NCRAS and HES APC all sources CPRD GOLD HES APC and ONS mortality CPRD GOLD alone HES APC alone Multiple malignant cancers recorded on the index date in CPRD GOLD or HES APC were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the NCRAS data if available and as multiple site cancer if not For each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upGold standard cancer case definitionWe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure Cancers recorded in NCRAS alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in HES APC alone or GOLD alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period Where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in NCRAS and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in NCRAS if there were more data sources in total with records for cancer at that site than data sources with contradictory recordsWe used NCRAS data to identify stage grade and treatment where available in the cancer registry only cohort Binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisStatistical analysisFor each cancer site and each individual or combined data source we combined our applied study definitions Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordWe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex We calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourcesWe used Kaplan Meier methods to describe mortality over time for cancers identified using each definition The ONS mortality death date was used for these analysesWe used the NCRAS only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timePatient public involvementPatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsRESULTSOf research quality patients in the CPRD GOLD January build were eligible for linkage to HES ONS mortality and NCRAS data in set were excluded due to unknown sex Of the remainder and had at least year of follow up between January and December and were included in the study population Using the gold standard algorithm incident cases of cancer were identified The number of patients identified with each cancer is presented in online supplementary appendix table Half n82 of these patients were male aged to aged to and aged or olderFigure presents PPVs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm When using NCRAS data alone to of cancers were confirmed by the algorithm for out of cancer sites the NCRAS only case definition gave the highest PPV Case definitions using data sources not including NCRAS generally had lower PPVs ranging from to for individual cancer sites For the four most common cancers breast lung colorectal and prostate PPVs were at least for all case definitions Minimal differences in PPVs were observed between age groups years and sexes online supplementary appendix figures Figure presents sensitivity values for each case definition Sensitivity was generally higher for the case definitions that included NCRAS data ranging from to for individual cancer sites except bladder cancer identified using NCRAS data alone and ¥ for the four most common cancers breast lung colorectal and prostate Sensitivity was also generally high for definitions using a combination of CPRD GOLD HES APC and ONS mortality data ranging from to ¥ for the four most common cancers Sensitivity was lower for case definitions that used CPRD GOLD alone range to for individual cancer sites or HES APC alone range to Sensitivity values for CPRD GOLD alone and HES APC alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures Post hoc analysis suggested that the low sensitivity of CPRD GOLD only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary an cancer codes n1108 in CPRD GOLD rather than contradictory information about the first cancer record n625 These incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 Bladder cancers that were not recorded in NCRAS data n3445 were commonly recorded in both HES APC and CPRD GOLD n2228 or in HES APC only with a subsequent unspecified or bladder cancer record in HES APC within months n995 Table describes the number of days median IQR and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used Case definitions using NCRAS alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using CPRD GOLD alone and HES APC aloneFigure describes mortality over time following incident cancer diagnoses ascertained from each case definition Minimal differences in mortality were observed between cancers identified from different case definitions Where variability was observed cancers identified using CPRD GOLD only had the lowest mortality rates eg kidney cancer and cancers identified using HES APC only or NCRAS only had higher mortality rates eg prostate cancer and bladder cancer respectivelyFigure describes completeness of grade and stage for cancers identified using NCRAS only Recording of grade was highly variable between cancers with gradual increases in completeness over time Completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in Post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure Online supplementary appendix figure describes recording of treatment modalities identified using NCRAS Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Positive Predictive Value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONSOffice of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0celitnecrep hthtRQI inadeMelitnecrepRQI inadeM hthtelitnecrep htht inadeMRQIelitnecrep hthtCPASEH DLOGDRPC ytil atromSNOdna CPASEH DLOGDRPC CPASEHdnaSARCN secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emTi l ebaT access recnaC lanoitaN SARCN atad eraC tneitaPdetti mdA scitsitatS edospEi latipsoH CPASEH knil ataDhcraeseR ecitcarP lacniilC DRPC metsys suovren lartnec SNCl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iiA setis recnac nommoc tsom ruoFDCiI nosrev sesaesDi fonoitacfissaCliscitsitatS lanoitaN rof ecfifO SNOi atadnoitartsgerrecnac ecvreS ssyanAdna liinoitartsgeRi lanoitanretnI eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaCi snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmuN ot ot ot ot ot Cl amoeym epitluMl ot Ci ameakueL ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtSARCN inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot C ytivac larOC laegahposeOC hcamotS CC latcerooClrecnaC amonaeml tnangilaMC saercnaPC gnuLC suretUC etatsorPC seiravOC yendKiC tsaerBCi xvreCCC SNCnarBiC reddaBlC amohpmyl s'inkgdo HnoNC idoryhTC revLiStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Mortality following first ever record of cancer in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphoma ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Completeness of grade and stage for cancers identified using NCRAS data only Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites Grading information is not applicable to brainCNS sarcoma or haematological cancers and not required by in the national data standard COSD for prostate cancer Core staging is not applicable to haematological and gynaecological cancers Other types of staging are recommended by COSD CNS central nervous system COSD Cancer Outcomes and Services Data set NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphomaStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access only Missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordedDISCUSSIONStatement of principal findingsWe investigated the use of different sources of electronic health record data to identify incident cancers For all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers Use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers Combining CPRD GOLD HES APC and ONS mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers Sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone Use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date Finally while we observed minimal changes in PPVs and sensitivities between and completeness of NCRAS cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 Completeness of cancer treatment recording was difficult to assess due to the absence of a missing categoryStrengths and weaknesses of the studyThe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from CPRD to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies We have also assessed the value of using NCRAS cancer registration data to measure stage grade and cancer treatment modalitiesA limitation of the study is that our gold standard algorithm is not validated We feel that we were justified in pre weighting NCRAS data as more reliable that other data sources as NCRAS is a highly validated data set that matches merges and quality checks data from multiple sources4 We did not consider NCRAS to be the outright gold standard as it is plausible that NCRAS does not identify all tumours diagnosed and treated in primary and secondary care For most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in HES APC CPRD GOLD or ONS mortality data but not in NCRAS These tumours may have been diagnosed and coded as invasive in primary or secondary care but not by NCRAS been incorrectly coded in HES APC CPRD GOLD or ONS mortality data not have been notified to NCRAS eg tumours treated in private hospitals or be the result of linkage errors between the data sets The proportion of cancers identified in HES APC but not in NCRAS is particularly high for bladder cancer This is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in NCRAS which are greatest for bladder cancer4 This explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in NCRAS compared with other data sources To identify incident cancers we required months of research quality follow up in CPRD GOLD prior to inclusion in the study Previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 The identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as NCRAS data collection started in HES APC in and ONS mortality data in and by the inclusion of all diagnostic codes in HES APC assuming that the first ever primary or secondary record identified incident cancer Reassuringly PPVs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases Requiring internal confirmation within months for cancers recorded in CPRD GOLD alone in our GOLD standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up Our data cut only included NCRAS data for the top cancers earlier cancers at other sites will have been missed in this studyIt is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesStrengths and weaknesses in relation to other studies discussing important differences in resultsThe most up to date study describing concordance between linked CPRD GOLD HES APC and NCRAS data sets demonstrated that to of the five most common cancers recorded in CPRD GOLD are not confirmed in either HES APC or cancer registration data and to of registered cancers are not recorded in CPRD GOLD8 For cancers recorded in both sources the diagnosis date was a median of to days later in CPRD GOLD than in the cancer registration data Using CPRD GOLD alone to identify these Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis Use of HES APC only identified a higher proportion of patients with the correct diagnosis date than CPRD GOLD but over represented older patients and those diagnosed through the emergency route The majority of registered cancers were picked up using both CPRD GOLD and HES APC ranging from for lung cancer to for breast cancer Previous research demonstrated similar results with substantial differences between cancer types5 Additionally a study using data from to found that using HES data in addition to NCRAS data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16Our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancersWe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards Levels of data completeness of staging information in the CPRD extract in were similar to those reported by the United Kingdom and Ireland Association of Cancer Registries UKAICR9Meaning of the study possible explanations and implications for clinicians and policymakersUse of NCRAS cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately Case definitions based on a combination of CPRD GOLD HES APC and ONS mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersThese findings | 2 |
" clinical trials have been conducted to clarify the beneficial effects of vd3 1α25dihydroxy vitamind3 also known as calcitriol treatment in prostate cancer however the molecular mechanisms underlying theseeffects are not fully understood recent studies on igfbp3 have indicated its intracellular functions in cell growthand apoptosis the aim of this study was to confirm the benefits of lowdose vd3 treatment and clarify themolecular mechanisms underlying these beneficial effects in prostate cancer cellsmethods the molecular effects of simultaneous treatment of lncap cells and their genetically modified cell lineswith low concentration of docetaxel and vd3 were biologically and biochemically analyzed to further determinethe effects of vd3 treatment on igfbp3 induction system cells were temporarily treated with vd3 in combinationwith a transcriptional inhibitor or protein synthesis inhibitor bcl2 protein and its mrna behavior were also observed inigfbp3 expressionmodified lncap cells to determine the involvement of igfbp3 in the suppression of bcl2 by vd3treatmentresults changes in igfbp3 expression levels in lncap cells indicated that it mediated the inhibition of cell growthinduced by vd3 treatment igfbp3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancercells to vd3 in combination with the anticancer drug we further identified the distinct property of the igfbp3induction system wherein temporal vd3 stimulationinduced prolonged igfbp3 expression and vd3 treatmentinduced increase in igfbp3 expression were optimized based on the protein concentration rather than the mrnaconcentration meanwhile bcl2 expression was downregulated by vd3 treatment in an igfbp3independent manner these findings indicate the molecular mechanisms of igfbp3 induction stimulated by vd3 and igfbp3independent bcl2 suppression by vd3 treatment in prostate cancer cells the results could prompt a reevaluation ofvd3 usage in therapy for patients with prostate cancerkeywords vitamin d nonlinear igfbp3 induction bcl2 suppression prostate cancer treatment correspondence satorusasagawatokushukaijp1molecular biology laboratory research institute nozaki tokushukai hospitaltanigawa daito osaka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cigarashi bmc cancer page of vitamin d has a central role in calcium and skeletalhomeostasis [ ] its pleiotropic role both in physiological and pathological phenomena such as cell growthimmune function and tumorigenesis has also been examined [] which revealed that exposure of cancercells to vitamin d significantly reduces the cell growthrate in multiple cancer types [] indeed recent epidemiologicalinvestigations have reported that highervitamin d concentration could prevent multiple types oftumorigenesis consistent with such finding for example an increase in colon cancer incidence with lowervitamin d dietary habits has been reported [ ]however suppressive effect on prostate cancer is stillunder discussion [ ]in turntraditional methodscurrently prostate cancer is one of the most commonthecancers in men worldwide clinical use ofprostatespecific antigen psatest dramatically improved the screening sensitivity of prostate cancer compared to that ofthenumber of patients with earlystage prostate cancer hasrapidly increased since the mid1990s [ ] unlikeother cancer types most cases of prostate cancer haveslow progression or have nonprogressive indolentsymptom and are localized in the prostate thus they areunlikely to cause poor physical condition or death therefore patients with prostate cancer need a less burdensome treatment in order to avoid potential harmfrom excessive treatmentalthough the impact of vitamin d as a single agent onprostate cancer has been investigated its significance remains under discussion [ ] meanwhile the synergistic or additive effects of vitamin d and its derivativeswith anticancer drugs on prostate cancer have beenclinically studied and encouraging results have been reported [ ] however the results of larger trials thatevaluated the synergistic effect of vitamin d in combination with docetaxel one of the firstline anticancerdrugs in prostate cancer chemotherapy showed limitedor nonsignificant benefit of vitamin d efficacy in castration or androgen deprivation therapyresistant prostatecancer [ ] furthermore overconsumption of 1α25dihydroxy vitamin d3 vd3 also known as calcitriolthe biologically active form of vitamin d3 from food orprolonged treatment with vd3 derivatives could triggerhypercalcemia resulting in physiological side effects therefore to date vd3 has not been proactivelyused in the treatment of patients with prostate cancerthe biological function of vitamin d is mainly mediatedby vitamin d receptor vdr which acts as a transcriptional factor vitamin d receptor elements vdreon the promoter region of target genes are recognizedand transcriptionally activated by vitamin dcoupledvdr consistent with the diverse physiological functionof vd3 vdre was identified not only in the gene related to calcium and skeletal homeostasis but also in thegene related to fundamental cellular functions includingcell growth igfbp3 is one of the families of sixhigh affinity igfbps and was originally found in plasmaas a stabilizer and transporter of igfs in the bloodstream interestingly vdre was found on the prothe igfbp3 gene and recent studies havemoter ofrevealed that igfbp3 functions inside the cell as wellregulating cell growth and apoptosis [ ]methodsthis study aimed to investigate igfbp3 induction byvitamin d treatment and determine its role in prostatecancer treatment with vitamin d in combination withanticancer drugs in order to provide molecular biologicalevidence of benefit of vitamin d and to suggest effectivevitamin d usage in prostate cancer treatmentchemicals and reagentsdihydrotestosterone dht and calcitriol vd3 purchased from tokyo chemical industry tokyo japanwere resolved in ethanol as a stock solution pei maxmolecular weight was purchased from polysciences pa usa the other chemicals and reagentswere purchased from wako pure chemicalosakajapan and sigmaaldrich st louis mo usaincluding testosteronecharcoal stripping of fetal bovine serum fbsfbs was purchased from gibco waltham ma usato deplete hormonesin fbsdextrancoated charcoal powder was added to theserum and the mixture was incubated with rotation at degree overnight thereafter the mixture was centrifugedto pellet charcoal and the supernatant was filtered througha 022μm polyvinylidene difluoride membrane thecharcoalstripped serum was used for all experimentstotalthe concentrations oftestosterone and totalvitamin d in the serum were determined using a totaltestosterone test kit abbott japan chiba japan and atotal vitamin d test kit roche basel switzerland according to manufacturers instructions the concentrations of total testosterone in the pre and posttreatmentserum were nm and less than nm limit of detection respectively the concentrations of total vitamin d in the pre and posttreatment serum were and nm respectively thus the basal concentrationsin the culture medium supplemented with fbs wereless than nm total testosterone and approximately nm total vitamin dcell culturethe lncap cell line was obtained from american typeculture collection and cultured in dulbeccos modified 0cigarashi bmc cancer page of eagle medium dmem sigmaaldrich supplementedwith 10charcoalstripping fbs the 293ft celllinewas purchased from invitrogen waltham ma usaand cultured in dmem supplemented with fbs mm of lglutamine sodium pyruvate and nonessentialamino acids the cells were cultured at a temperature of °c in co2humidified condition the mycoplasma contamination was routinely checked and confirmed as negativecell growth assaydmem supplemented with charcoalstripping fbs was used for the cell growth assay the cells were seededat à cells per well in a 6well plate the next daythe medium was replaced with ml of fresh mediumand nm dht andor nm vd3 were added the cellculture was continued throughout the indicated periodthe cultured cells were trypsinized and the number ofcells was assessed using an automated cell countercountess iitm fl invitrogen each assay was repeatedat least three times and similar results were obtainedwestern blottingthe antibodies used for western blotting were mouseantiigfbp3 santa cruz biotechnology mouseantiβactin santa cruz biotechnology mouseantibcl2 santacruz biotechnology and horseradish peroxidaseconjugated secondary antibodies jackson immunoresearch laboratories westgrove pa usa the collected cells were resuspendedin ripa buffer supplemented with protease and phosphatase inhibitors roche basel switzerland and lysedusing bioruptortm ii sonicator cosmo bio tokyojapan cell lysates were resolved by nupagegels invitrogen and transferred onto polyvinylidenefluoride membrane millipore burlington ma usathe signals were developed using enhanced chemiluminescence reagent perkinelmer waltham ma usaand luminograph i atto tokyo japan was used forimage capture quantification of band signal wasanalyzed using cs analyzer software atto at leasttwo biological replicates of each experiment were performed with similar resultsrealtime reverse transcriptionpolymerase chain reactionthe total rna from the cultured cells was extractedusing trizol reagent invitrogen according to the manufacturers instruction the rna was reverse transcribedby the primescript rt reagent kit takara bio shigajapan using oligodt quantitative reverse transcriptionpolymerase chain reaction rtpcr reaction wasperformed using tb green premix ex taq ii takaraand genespecific primers supplementary table bycfx96 touch realtimepcr system bioradlaboratories hercules ca usa gene expression datawere normalized against glyceraldehyde 3phosphate dehydrogenase or hprt1 as internal control at leastthree biological replicates of each experiment were performed and similar results were obtainedflow cytometric analysis of cell cycle and apoptosisfor cell cycle analysis and apoptotic cell detection flowcytometric analysis was performed using the guavaeasycyte plus flow cytometry system millipore andguava cell cycle reagent and annexin v fitc apoptosiskit millipore according to manufacturers instructionas previously described at least three biologicalreplicates of each experiment were performed and similar results were obtainedlentiviral construction and transductionbackbone vectors plko1 puro plasmid andplenti cmv puro dest w118 plasmid were provided by drs bob weinberg eric campeau andpaul kaufman respectively via addgene ref pentr1a plasmid and lentiviral packaging mix plp1plp2 and plpvsvg were purchased from invitrogenfulllength igfbp3 was cloned from lncap cell complementary dna cdna using kod fx neo toyoboosaka japan with a specific primer set supplementarytable and inserted into the pentr1a vector thenthe sequence was confirmed the igfbp3 cdna was introduced into the plenti cmv puro dest vector by recombinaseenzymeinvitrogen to generate the lentiviral expression vectorspecific short hairpin rnas shrnas were designedusing invitrogen or biosettia websites the selectedtarget sequence oligos supplementary table wereannealed and inserted into the plko1 puro vector according to addgenes instruction the lentiviral expressionvector or shrna vector was cotransfected with the lentiviral packaging mix into the 293ft cells using pei maxinstead of lipofectamine according to invitrogensinstruction twentyfour hours posttransfectionthemedium was replaced with the culture medium forlncap cells one day later the lentiviruscontaining supernatants were collected and filtrated through a 045mmpolyvinylidene fluoride filter milliporereaction using lr clonaseiilentivirus infectionone day before infection à lncap cells wereplated into a 10cm dish then the culture medium wasreplaced with the lentiviruscontaining supernatant andculture was continued twentyfour hours post infectionreplaced with fresh culturemedium two days later the medium was replaced withfresh culture medium that contained μgml of puromycin and culture was continued until the noninfectedthe medium was 0cigarashi bmc cancer page of control cells were completely killed the puromycinselected cells were then subjected to each assaystatistical analysisresults are presented as mean ± standard deviation unless otherwise specified pairs of groups were comparedusing a twotailed unpaired students ttest onewayanalysis of variance was used for multiplegroup comparisons rather than specifying three a pvalue was considered statistically significant all statistical analyses were performed using excel software microsoftredmond wa usa and statcel3 addin for exceloms publishing tokyo japanresultsvd3 reduces cell growth rateconsistent with a previous report that treatmentwith vd3 inhibits growth of prostate cancer cells ourresults showed that vd3 treatment reduced the cellgrowth rate in a dosedependent manner and nm fig 1a left as shown below igfbp3 induction activity of vd3 was reached to plateau at nm concentration fig 4a on the other hand testosterone hasbeen reported to stimulate the growth of prostate cancer and the results of this study confirmed that dhttreatment from very low concentration nm stimulated cell growth rate and its activity was reached toplateau at less than nm concentration fig 1a centerthe purpose of our study was to investigate the role ofvd3igfbp3 induction system in cell growth inhibitionand to propose the potency oflowdose vd3 usagewhich could evade sideeffect of vd3 treatment such ashypercalcemia in therapy for patients with prostate cancer thus nm of dht and nm of vd3 concentrations were chosen as minimum but stably workingconcentration for following experiment previouslyithas been demonstrated that simultaneous treatmentwith vd3 and dht enhanced the reduction of cellgrowth rate compared to treatment with vd3 alone anda similar result was reproduced with lowdose dht nm and vd3 nm in this study fig 1a right tofurther characterize growth inhibitory effect with lowdose of dht nm and vd3 nm cell cycle andapoptotic analyses were performed with flow cytometrythe cell cycle analysis revealed that there was no significant change in the cell cycle phase distribution amongfig cellular response of lncap cells treated with vd3 and dht a effect of combined treatment of vd3 and dht on cell growth in lncapcells left vd3 treatment reduced cell growth in a dosedependent manner center dht treatment increased cell growth at lowerconcentrations right simultaneous treatment of vd3 and dht enhanced the reduction of cell growth compared to treatment with vd3 aloneb change in cell cycle phase by vd3 or dht treatment neither vd3 nor dht treatment significantly changed the cell cycle phase c induction ofapoptosis by vd3 or dht treatment neither lowdose vd3 nor dht treatment influenced apoptosis at shortterm d induction of igfbp3expression by vd3 or dht treatment vd3 treatmentinduced igfbp3 expression and cotreatment with dht enhanced the expression level ofigfbp3the ratio indicates the density of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium condition the uncropped fulllength blot images are presented in supplementary fig 5a 0cigarashi bmc cancer page of control no treatment dht nm vd3 nm anddhtvd3 treatment conditions fig 1b suggestingthat lowdose of vd3 or dhtvd3 treatment did notarrest the cell cycle at a specific phase previous reportshave shown that longterm vd3 treatment has apoptosisinducible activity and dht has apoptosis inhibitingactivity in a dosedependent manner in lncapcells however it was unclear or controversial whetherlowerconcentration of vd3 and dht at shorttermcould influence apoptosis respectively in lncap cellsthus the apoptosis assay was performed with nm ofdht and nm of vd3 for shortterm and found thatneither lowerdose dht vd3 nor dhtvd3 treatmentfor shortterm influenced apoptosis fig 1c these results suggested that the decrease in the cell number induced by lowdose vd3 or dhtvd3 treatment wasmainly due to a decrease in the cell growth rate to further address what was occurring at the molecular levelduring lowdose dht andor vd3 treatment the genesknown to regulate the cell cycle and inducible by dhtandor vd3 treatment were chosen and messengerrna mrna induction was quantitatively measuredfig 1d upper supplementary fig 1a the quantitativertpcr results showed that igfbp3 mrna inductionwas positively correlated to cell growth suppression inresponse to lowdose vd3 or dhtvd3 treatment andthe expression strength was dramatically sensitive tovd3 or dhtvd3 treatment consistent with thatigfbp3 protein was markedly induced by vd3 treatment and it was enhanced by simultaneous treatment ofvd3 with dht a similar response was observed in theexpression of ar the receptor of dht which wasknown to a one oftarget of vdr supplementaryfig 1bmultiple recent studies have revealed that igfbp3functions in cellular response including cell growth andapoptosisin an insulinlike growth factor igfindependent manner considering these findings we believethat igfbp3 can be a key molecule for vd3 treatmentin prostate cancer cellsigfbp3 was a dominant factor in cell growth suppressionto confirm ifigfbp3 dominantly suppresses cellgrowth in lncap cells we applied the gainoffunctionand lossoffunction approach using a lentivirus systemfirst we generated igfbp3overexpressing lncapcells and found that the expression of igfbp3 mrnawas about higher compared to that by lowdosedhtvd3 treatment fig 2a as an infection controlegfpoverexpressing lncap cells were also generatedand it was confirmed that lentivirus infection per se didnot induce igfbp3 expression using these cell linesthe effect of igfbp3 on cell growth was observed fig2b results showed that the cell number of egfpoverexpressed cells treated with nm of dht and nm of vd3 for days was decreased to comparedwith that of untreated cells and the igfbp3overexpressing cells showed comparable cell growth decreasewithout dhtvd3 treatment next we generatedshrna for igfbp3 shigfbp3expressing lncap cellsthe knockdown of igfbp3 mrna and protein inducedby lowdose dhtvd3 treatment was confirmed inshigfbp3expressing lncap cells fig 2c using thiscell line the effect of lowdose dht and vd3 treatmenton cell growth was observed as we expected the suppressive efficacy of lowdose vd3 on cell growth wasweakening and simultaneous treatment with nm ofdht and nm of vd3 increased cell growth fig 2dtaken together these data indicated igfbp3dominantfactor of cell growth suppression induced by lowdosevd3 treatment in lncap cellsacceleration of anticancer drug effect by vd3as previously reported and we demonstrated abovevd3 alone is not cytotoxic at physiological and pharmacological concentrations meanwhile simultaneous treatment with vd3 has been reported to improve theefficacy of anticancer drugs including docetaxel howeverits molecular mechanisms were remained not fully uncovered here we supposed that igfbp3 might be amediator of vd3induced sensitization to anticancerdrugs in prostate cancer cells to confirm this hypothesis lncap cells were treated with lowdose of dhtvd3 in combination with several concentrations of docetaxel to determine the appropriate concentration ofdocetaxel for evaluating the vd3 effect we first screenedthe concentration of docetaxel based on cytotoxic activity and found that a docetaxel concentration nmkilled bulk of the cells treated fig 3a here ic50 ofdocetaxel was nm in our assay and it was consistent with previous reports nm thus a docetaxel concentration nm was chosen to observe theeffect of combinatoriallowdose dhtvd3 treatmentfor the following assay to evaluate the synergistic effectof dhtvd3 on cytotoxicity by docetaxel lncap cellswere treated with or nm of docetaxel withor without dhtvd3 and results showed that lowdosedhtvd3 with docetaxel reduced the living cell numberat the concentration range of nm but the effectwas masked when nm docetaxel was applied fig 3bsimilarlylowdose dhtvd3 with cisplatin reducedthe living cell number at the concentration range of nm supplementary fig to see if these enhancedcytotoxicity effects were dependent on igfbp3 igfbpoverexpressed or shigfbp3expressed lncap cellswere analyzed in the same manner indeed in igfbp3overexpressed cells the living cell number was reducedby docetaxel without dhtvd3 addition fig 3c 0cigarashi bmc cancer page of fig igfbp3 mediates the effect of vd3 on cell growth in lncap cells a overexpression of igfbp3 in lncap cells lncap cells were infectedwith lentivirus containing the igfbp3 gene and its overexpression was confirmed by quantitative reverse transcriptionpolymerase chainreaction b suppression of cell growth by igfbp3 igfbp3overexpressed cells were cultured as they were and control cells were cultured withdhtvd3 for days and then the cell number was measured c knockdown of igfbp3 in lncap cells lncap cells were infected with lentiviruscontaining shrna for igfbp3 and igfbp3 knockdown was confirmed by quantitative reverse transcriptionpolymerase chain reaction andwestern blotting the ratio indicates the density of igfbp3 band normalized by corresponding βactin band d the igfbp3 knockdown cells weretreated with dht andor vd3 for days and then the cell number was measured the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5bcorrespondingly in the shigfbp3expressed cells reduction of living cell number by dhtvd3 addition wascanceled rather the cell living number was increasedliving cell number despitefig 3d the increase ofdhtvd3 addition in docetaxeltreated shigfbp3expressed cells was assumed by cancelation of vd3 effect and emerging of dht effect on cell growth basedthese findings lowdose dhtvd3induced enhancedcytotoxicity by docetaxel on lncap cells was dependenton igfbp3 expressioncharacterization of the igfbp3 induction mechanismas demonstrated above igfbp3 had a pivotal role inlowdose dhtvd3induced enhanced cytotoxicity byantitumor drugs to further dissect the igfbp3 induction mechanism and to provide the molecular evidenceof vd3 treatment for clinical research mechanisms ofigfbp3 induction by dht and vd3 were analyzed aspreviously reported in prostate cancer cells vd3 treatment induces cyp24a1 as well an enzyme that catalyzes vd3 to its inactive form as a negative feedbackfactor the induced cyp24a1 limits the efficacy of vd3meanwhile activated ar induced by dht treatmentsuppresses cyp24a1 transcription thus cancelling thenegativefeedback loop to inactivate vd3 consistentwith that cyp24a1 induction by vd3 treatment and itssuppression by simultaneous treatment with dht wereconfirmed even when we applied lowdose dht and 0cigarashi bmc cancer page of fig vd3 enhanced cytotoxicity of docetaxel a dosedependent cytotoxicity of docetaxel lncap cells were treated with docetaxel for daysand then the living cell number was measured ic50 was nm b simultaneous treatment of vd3 with dosedependent docetaxelsimultaneous treatment of vd3 with docetaxel reduced the living cell number at nm range c igfbp3 overexpression was conducive todhtvd3 treatment on docetaxel treatment igfbp3overexpressed or control cells were treated with dhtvd3 and nm of docetaxel andcultured for days and then the cell number was measured d igfbp3 knockdown canceled the effect of vd3 on docetaxel treatment in igfbp knockdown cells dhtvd3 treatment increased the cell number compared to that of the notreatment control and the cell number wasalmost equal to that of the dht treated sample the concentration of docetaxel used was nm the all experiments were performed in serumcontaining medium conditionvd3 which were enough to induce and suppresscyp24a1 expression supplementary fig meaningthat h lowdose dhtvd3 treatment could cancel thecyp24a1driven negativefeedback loop to further dissect the mechanism of igfbp3 expression in lncapcells the cells were treated with vd3 alone or dhtfixed in nm and vd3 in a dosedependent manner and nm when treated with vd3alone the induced igfbp3 reached a plateau at to nm in contrast when treated with vd3 together withdht the amount of induced igfbp3 was increased according to the increment of vd3 concentration fig 4athese results indicated that lowdose dht could improve igfbp3 induction activity of vd3 throughcyp24a1 suppressionclinically highdose vd3 or its derivatives for treatment can cause hypercalcemia thus its continual usageshould be carefully monitored to avoid sideeffect ofvd3 here we wondered if continual vd3 treatmentfig 4btop herewould be required for maintaining igfbp3 induction inprostate cancer cells to address this lncap cells weretreated with vd3 alone or lowdose dhtvd3 for or days followed by washout which was done by replacing the culture medium and continuing the culturefor days in totalintracellularigfbp3 protein was observed by western blottinginterestingly 1day treatment of vd3 or dhtvd3 induced stable igfbp3 expression fig 4b bottom although treatment of vd3 alone showed mild igfbp3induction compared to that by dhtvd3 note thatigfbp3 showed similar strength of expression between and days of vd3 or dhtvd3 treatment this resultclearly indicated that temporal vd3 treatment couldinduce prolonged stable igfbp3 expressionthis nonlinear response suggested the presence of aunique molecular property underlying the igfbp3 expression mechanism generally nonlinear cellular response such as sustained protein expression by temporal 0cigarashi bmc cancer page of fig characterization of igfbp3 expression induced by vd3 and dht treatments a effect of dosedependent vd3 treatment with or withoutdht on igfbp3 induction lncap cells were treated with vd3 alone at the indicated concentration or with vd3 and nm dht top westernblotting image of igfbp3 and bottom quantified graph of igfbp3 induction open circles vd3 alone filled circles dht vd3 b effect oftemporal treatment of dhtvd3 on igfbp3 induction top schematic time course of temporal treatment of vd3 and bottom western blottingimage of igfbp3 the ratio indicates the density of igfbp3 band normalized by corresponding βactin band c effect of mrna transcription andprotein synthesis on the stability of igfbp3 expression induced by dhtvd3 lncap cells were treated with vd3dht for day followed byactinomycin d actd μm or cycloheximide chx μm for another day after vd3dht was washed out or left as is top quantification graphof igfbp3 mrna induction middle western blotting image of igfbp3 and the combination of treatments bottom the ratio indicates thedensity of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium conditionthe uncropped fulllength blot images are presented in supplementary fig 5ctriggered by positivefeedback loopstimulation wasmechanismin which protein synthesis and transcriptional regulation was included as hysterical responsedriver thus to further dissect if protein synthesis ortranscriptional regulation or both are involved in nonlinear vd3igfbp3 induction actinomycin d and cycloheximide which are transcriptionalinhibitor andprotein synthesis inhibitor respectively were added withvd3 or dhtvd3 and behavior of igfbp3 proteinand its mrna was observed experimentally actinomycin d or cycloheximide was added day after treatmentwith vd3 alone or dhtvd3 and the culture was continued for another day when interfered with μm ofactinomycin d there was no change in igfbp3 mrnaor protein expression fig 4c by contrast when interfered with μm of cycloheximide the igfbp3 proteinwas immediately reduced howeverthe mrna wasunexpectedly increased several times higher than thosewithout cycloheximide interference fig 4c the unexpected mrna increase became stronger when dhtvd3 washout was performed ahead of the cycloheximideinterference these cellular responses on igfbp3 induction interfered by actinomycin d and cycloheximidesuggested that the cells had a protein abundancebasedpositivefeedback loop to maintain the total amount ofigfbp3 via transcriptional controligfbp3independent bcl2 suppression by vd3as shown above although lowdose dht andor vd3treatment did not induce apoptosis fig 1d vd3 treatment rendered lncap cells sensitive to the antitumordrugs fig 3b and supplementary fig suggesting thatany apoptosisrelated factor might be influenced to addressinvestigated the behavior ofidea wethis 0cigarashi bmc cancer page of apoptosisrelated molecules in response to lowdosevd3dht treatment consistent with previous report although the concentration of vd3 was higher thanthat we used here bcl2 protein an antiapoptotic molecule was downregulated by lowdose vd3 treatmentfig 5a compared to that by vd3 alone it seemed thatbcl2 downregulation by dhtvd3 was not seemed tobe enhanced unlike to igfbp3 expression suggestingthat bcl2 downregulation by vd3 was igfbp3 induction independent manner to see whether bcl2 downregulation was igfbp3dependent or not the behaviorof the expression of bcl2 protein and mrna was observed in shigfbp3expressedcells interestingly despitethe igfbp3 disappearance bcl2 downregulation wasobserved according to vd3 or dhtvd3 treatmentfig 5b bottom and it was not significantly different atinigfbp3expressionenhancedprotein and mrna expression level compared to that inshctrlexpressing cells moreover in order to confirmthatconditionshcyp24a1 expression cells were established in whichvd3 effect on igfbp3 induction was expected tostrengthen the knockdown of cyp24a1 under the vd3treatment condition was confirmed by qrtpcr supplementary fig indeed the amount of igfbp3 protein wasincreased in shcyp24a1expressing cellscompared to that in shctrl cells fig 5c bottom despiteigfbp3 expression downregulation bcl2 protein by lowdose vd3 or dhtvd3treatment was not observed compared to that in shctrlcells fig 5c bottom also the expression of bcl2mrna was not significantly changed fig 5ctopthese results suggested that the downregulation of bclenhancement offig igfbp3independent reduction of bcl2 protein expression induced by vd3 a western blotting image of bcl2 reduction by vd3treatment the ratio indicates the density of bcl2 band normalized by corresponding βactin band b effect of igfbp3 suppression on bcl2reduction by vd3 treatment the cells were infected with lentivirus encoding of shrna for igfbp3 and then treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band c effect of igfbp3 overexpression on bcl2 reduction byvd3 treatment the cells were infected with lentivirus encoding of the cyp24a1 gene then the cells were treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5d e 0cigarashi bmc cancer page of protein by lowdose vd3 treatment was independentof igfbp3 induction besides the mrna of bcl | 0 |
"Sulindac is a ligand of the aryl hydrocarbon receptor (AhR) an xenobiotic-sensing nuclear receptor that can be activated by chemical structures containing planar aromatic hydrocarbons and thus evokes a cellular response that to detoxify xenobiotics. AhR activation leads to transcriptional upregulation of the NQO1 gene [62] [63]. In previous studies [30] [64] [65] sulindac and its two metabolites have been used to treat cancer cells at concentrations of 200 µM 1 mM i.e. the concentrations used in our present study. In addition to reducing the growth of polyps all three increase NQO1 activity and expression in colon cancer cells [28] and might therefore be good candidates to increase the cytotoxic effect of ?-lapachone against lung cancer cells. When two cancer cell lines CL1-1 and CL1-5 with low NQO1 expression and activity were co-incubated with sulindac or its metabolites and ?-lapachone much higher cell death was seen with the CL1-5 cells than the CL1-1 cells ( and 7). These results demonstrated that the effect of sulindac and its metabolites in upregulating NQO1 was greater in CL1-5 cells which has lower NQO1 level and activity than CL1-1 cells showing that sulindac and its metabolites can be used to increase the ?-lapachone sensitivity of cells with lower NQO1 levels. Many other compounds such as toxifolin [32] and resveratrol [66] can increase NQO1 expression or activity but are not FDA-approved. A search is underway for other compounds that can increase the activity or expression of NQO1 using high-throughput library screening and two compounds DMEBP and TRES were recently found to be potent NQO1 inducers with low toxicity [3]. These compounds may also be valuable in increasing ?-lapachone cytotoxicity for cancer cells with low NQO1 expression or activity. The NQO1 Inhibitor Dicoumarol or Transfection with NQO1 siRNA Inhibits the Effect of Sulindac on ?-lapachone Toxicity for Lung Cancer Cells Dicoumarol is widely used as a specific pharmacologic inhibitor of NQO1 and has been shown to inhibit both enzyme activity and expression [45] [67] [68]. NQO1 siRNA designed to specifically target NQO1 mRNA can lower the expression of NQO1 mRNA and protein. In our study both agents blocked the synergistic effect of sulindac or its metabolites and ?-lapachone on decreasing the survival of CL1-1 or CL1-5 cells. Although ?-lapachone is very toxic for many cancer cells cells with lower NQO1 levels are less sensitive. However from the present study we can conclude that sulindac and its metabolites increase NQO1 expression and enzyme activity and thus are potential synergistic drugs that might be used in combination with ?-lapachone to treat cancer cells with high resistance to ?-lapachone cytotoxicity. Supporting Information Figure S1 ?-lapachone causes cell death of CL1-1 and CL1-5 cells by decreasing the mitochondrial membrane potential. (A) Cells were left untreated or were incubated with 5 µM ?-lapachone for the indicated time and then the cell cycle distribution was analyzed using propidium iodide staining and flow cytometry. (B) Cells were incubated with 5 µM ?-lapachone for the indicated time then pro-caspase 3 and caspase 3 levels were analyzed by Western blotting. (C) Cells were incubated with 5 µM ?-lapachone for the indicated time then the mitochondrial membrane potential (MMP) was measured using the dye JC1 (Life Technology) and flow cytometry. (D) Cells were incubated with 5 µM ?-lapachone for the indicated time and then intracellular H2O2 levels were measured. (TIF) Click here for additional data file. Figure S2 zVAD-FMK ALLM and ALLN do not block the cytotoxicity of ?-lapachone. CL1-1 cells (A) or CL1-5 cells (B) were left untreated or were incubated for 1 h with the indicated concentration of the pan caspase inhibitor zVAD (left panels) or the calpain inhibitor ALLM (center panels) or ALLN (right panels) then 5 µM ?-lapachone was added for 12 or 24 h and cell viability measured using the MTT assay and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S3 Dicoumarol an NQO1 inhibitor inhibits NQO1 activity and blocks the increase in intracellular calcium levels induced by ?-lapachone. (A) CL1-1 cells (left) or CL1-5 cells (right) were left untreated (CTL) or were incubated with 10 µM dicoumarol for 6 h then NQO1 activity was measured. (B) CL1-1 cells (top panel) or CL1-5 cells (bottom panel) were left untreated or were incubated with 10 µM dicoumarol and/or 5 µM ?-lapachone for 1 h then were stained with Fluo-4 and the intensity of the Fluo-4 fluorescence measured by flow cytometry. (TIF) Click here for additional data file. Figure S4 Sulindac and its metabolites do not affect survival of lung cancer cells. CL1-1 CL1-5 or A549 cells were left untreated or were incubated for 54 h with 100 or 250 µM sulindac (left panel) or sulindac sulfone (center panel) or for 12 h with 100 or 250 µM sulindac sulfide (right panel) then cell survival was measured by the MTT assay and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S5 The cytotoxic effect of ?-lapachone on A549 cells is enhanced by sulindac and its metabolites. Two sets of cells were left untreated or were incubated for 6 h with the indicated concentration of sulindac sulindac sulfone or sulindac sulfide then 2 µM ?-lapachone was added to one set and incubation continued for 12 h when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. (TIF) Click here for additional data file. Figure S6 NQO1 siRNA has no effect on cell morphology or cell growth. CL1-1 cells (top) and CL1-5 (bottom) were transfected with negative siRNA or NQO1 siRNA for 1 to 3 days then pictures were taken using a digital camera and phase contrast microscopy. The scale bar represents 50 µm. (TIF) Click here for additional data file. Figure S7 NQO1 RNA levels are decreased by siRNA targeting NQO1. A549 CL1-1 or CL1-5 cells were transfected for 48 h with siRNA targeting NQO1 (siNQO1) or control siRNA (siNeg) and then NQO1 mRNA levels were measured by realtime PCR and expressed as a fold change compared to the value for CL1-5 cells transfected with siNeg. * : p<0.05 compared to the result for the corresponding siNeg-transfected cells. (TIF) Click here for additional data file. Table S1 Primers used in the realtime PCR for actin and NQO1. (TIF) Click here for additional data file. Materials and Methods S1 (DOCX) Click here for additional data file. This work was supported by grants (NSC 101-2320-B-002-020-MY3 NSC 98-2320-B-715-001-MY3 (YPC) and NSC 101-2320-B-002-008) from the National Science Council Taiwan. References 1 PardeeAB LiYZ LiCJ (2002) Cancer therapy with beta-lapachone. Curr Cancer Drug Targets2: 22724212188909 2 TagliarinoC PinkJJ ReinickeKE SimmersSM Wuerzberger-DavisSM et al (2003) Mu-calpain activation in beta-lapachone-mediated apoptosis. Cancer Biol Ther2: 14115212750552 3 TanXL MarquardtG MassimiAB ShiM HanW et al (2012) High-throughput library screening identifies two novel NQO1 inducers in human lung cells. Am J Respir Cell Mol Biol46: 36537122021338 4 MinamiT AdachiM KawamuraR ZhangY ShinomuraY et al (2005) Sulindac enhances the proteasome inhibitor bortezomib-mediated oxidative stress and anticancer activity. Clin Cancer Res11: 5248525616033843 5 TeraiK DongGZ OhET ParkMT GuY et al (2009) Cisplatin enhances the anticancer effect of beta-lapachone by upregulating NQO1. " | 1 |
"peroxisome proliferatoractivated receptorsppars asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms pparalpha ppar betadelta and ppar gamma ppars formheterodimers with retinoic x receptors and regulate theexpression of various genes upon ligand binding ppars alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes ppars asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions for instance ppar alpha also functions in fatty acidbetaoxidation and vascular ammation ppar gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes ppar betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression ppar alpha and ppar gamma exert predominantly anantiangiogenic eï¬ect [] but there still exist conï¬ictingstudies showing opposite results [ ] on the contraryppar betadelta produces more obviously proangiogeniceï¬ects [] in this review we will focus on the promotingrole of ppar betadelta in angiogenesis especially in tumorangiogenesis the network of interplay between ppar betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identiï¬ed as direct ppar betadelta target genes angiogenesisangiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells epc migrate to sites of vascularization then diï¬erentiate into endothelial cells ec andcoalesce into the initial vascular plexus [ ] besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cppar researchmmps extracellular matrix ecm and basement membrane componentsangiogenesis is a physiological and vital process in development and growth an imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growththus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an angiogenicswitch of tumor cells is turned on during tumor progression the angiogenic switch is often activated and remainson [] inducing angiogenesis is known as a hallmarkof cancer and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones tumor angiogenesis tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis the tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningtherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread thus this has been regarded as a critical target forantitumor therapy ppar alpha and angiogenesisit was reported ï¬rstly that a selective ppar alpha agonistwy14643 did not show any eï¬ect on angiogenesis or ec proliferation but some subsequent studies showed that theactivation of ppar alpha inhibited angiogenesis in vitro byusing fenoï¬brate a clinically used ppar alpha agonist moreover fenoï¬brate suppressed ec proliferation migration and tube formation through inhibition of protein kinaseb akt and disruption of the cytoskeleton furthermore ppar alpha activation was shown to inhibit vascularendothelial growth factor vegf induced ec migrationand basic ï¬broblast growth factor bfgffgf2 inducedcorneal angiogenesis in vitro and in vivo especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma lewis lungcarcinoma llc ï¬brosarcoma and glioblastoma due to asystemic treatment of ppar alpha ligand and the antiangiogenic state induced through activation of ppar alpha withelevated thrombospondin1 tsp1 and endostatin expression howeverit was demonstrated inanother observation that activation of ppar alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase enos and akt via avegfdependent manner furthermore zhang andward also suggested that ppar alpha activation inducedproangiogenic responses in human ocular cells inanother study it was shown that a new ppar alpha agonistrk13675 had no eï¬ect on angiogenesis recentlyppar alpha activation is further shown to have antineovascularization eï¬ects with downregulation of vegf and angiopoietin expression in a rat alkali burn model in summary the role of ppar alpha in angiogenesis isstill controversial some observations showed that ligandactivation of ppar alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as tsp1 and endostatin or downregulation of proangiogenic factors including vegf fgf2 akt and angiopoietins others also reported opposite results showing aproangiogenic role upon ppar alpha activation thus thespeciï¬c molecular mechanism is still unclear and needs tobe further studied ppar gamma and angiogenesisligand activation of ppar gamma was previously shown toinhibit human umbilical vein endothelial cell huvec tubeformation in collagen gels and vegfinduced choroidalneovascularization in vitro and in vivo another studyalso demonstrated that ec apoptosis was induced throughtreatment with the ppar gamma ligand 15dpgj2 furthermore rosiglitazone a potent ppar gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bfgfinduced corneal neovascularizationin vivo moreover a similar observation also displayedthe inhibition of vegfinduced angiogenesis in a chickchorioallantonic membrane model in a mouse modelwith ischemiainduced retinopathy pioglitazone a ppargamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of antiammatory adipokine adiponectin additionally theppar gamma antagonist gw9662 was shown to reverseomega3 polyunsaturated fatty acidinduced reduction ofeselectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 implicatingan antiangiogenic potential of ppar gamma itself howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of epc in vitro andin vivo via a phosphoinositide3kinase pi3k dependentmanner nadra observed that ppar gammanull embryosdisplayed a vascular structural defect at e95 moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the ppar gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingvegf proliferin and plateletendothelial cell adhesionmolecule1 pecam1cd31 suggesting a crucial roleof ppar gamma in placental vascular development the 0cppar researchmajor antiangiogenic properties on ppar gamma activationwere also reviewed here notablyin most cancersthe canonical wntbetacatenin pathway is upregulated while on the contrary ppargamma is downregulated interestingly in numerous tissuesthe activation of ppar gamma inhibits the betacateninpathway whereascanonicalwntbetacatenin signal cascade also inactivates ppargamma implicating a negative regulatory role of ppargamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthethein summary ppar gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of vegf or bfgfinduced neovascularization andreduction of the expression level of some proangiogenicfactors ppar betadelta and angiogenesisunlike ppar alpha and ppar gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof ppar betadelta on physiological and pathological angiogenesis the ï¬rst evidence provided in a study is that activation of ppar betadelta with gw501516 a highly selectiveppar betadelta agonistinduces huvec proliferationand an increased expression of vegf and its receptorvegfr1 flt1 besides inducing ec proliferationppar betadelta activation by itsligand prostacyclinpgi2 also stimulates upregulation of alpha expression an antiapoptotic and antiammatory protein whichthereby protects ecs from h2o2induced apoptosis and oxidant injury moreover a subsequent study further provides evidence that activation of ppar betadelta withgw501516 induces angiogenesis during which vegfrelease is considered as a major trigger factor ï¬rstlysuggesting the promotion for angiogenesis upon pparbetadelta activationmüllerbrüsselbach show that ppar betadelta mice implanted with llc and b16 melanoma exhibit diminished blood ï¬ow and immature microvascular structurescompared with wildtype mice moreover reexpression ofppar betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization indicating a crucial role of ppar betadelta in tumorvascularization additionally another study also observedreduced levels of calcium intracellular channel protein clic4 but it observed enhanced expression of cellular retinol binding protein crbp1 in migrating ecs from pparbetadeltanull mice both of which play a role in tumorvascularization [ ] it was reported that ppar betadeltawas required for placentation and most of the pparbetadeltanull mutant embryos died at e95 to e105 due toabnormal celltocell communication atthe placentaldecidual interface however in these studies [] adefect in angiogenesis was not observed during normal development in ppar betadeltaknockout micesome observations also show the important role of pparbetadelta in physiological angiogenesis for instance skeletal musclespeciï¬c ppar betadelta overexpression leads toppar betadeltaan increase in the number of oxidative muscle ï¬bers andrunning endurance in adult mice [] moreover pparbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective ppar betadelta agonistgw0742treated animals furthermore in the heartpharmacologicalstimulation withgw0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin interestingly the same cardiac phenotype wasalso observed after treatment with the ppar betadelta agonist gw501516implicating a response speciï¬city forppar betadelta stimulation calcineurin activationfurther leads to the stimulation of nuclear factoractivatedt cell c3 nfatc3 and an enhanced expression of hypoxiainducible factor alpha hif1alpha and cyclindependentkinase cdk9 overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinppar betadelta may act as a key regulator in mediatingpathological angiogenesis for instance ppar betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an angiopoietinlike protein angptl4 dependent manner implicating the potential of pparbetadelta in modulating pathological ocular angiogenesisrecently an observation reported that ppar betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andppar betadelta promoted laserinduced choroidal neovascular cnv lesions in ppar betadelta mice moreover pharmacological inhibition of ppar betadelta with theantagonist gsk0660 also resulted in a signiï¬cantly decreasedcnv lesion size in vivo suggesting a functional role of pparbetadelta in the development of cnv lesions this indicates that ppar betadelta has an important association withpathological angiogenesisangiotensin ii ang ii the biologically active peptide ofthe reninangiotensin system ras is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction inexperimental systemsthe antitumor eï¬ects of diverseace inhibitors show that these inhibit cell proliferationand possessand antiammatory eï¬ects [] it has been shown recentlythat activation of ppar betadelta inhibits ang iistimulated protein synthesis in a concentrationdependentmanner and suppresses ang iiinduced generation of reactive oxygen species ros in vascular smooth muscle cells ppar betadelta was further shown to inhibit angiimediated atherosclerosis however it is not clearuntil now if ppar betadelta activation can be consideredis foras an ace inhibitormimicking approach as itexample the case for ppar gamma activators antiangiogenicantimetastatic 0cppar researchthe relevance offurthermorethis hypothetical pparbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologybesides inducing angiogenesis it has been demonstratedthat ppar betadelta directly acts on early epc through activation of the akt pathway and induces an enhanced vasculogenesis similarlythe ppar betadeltamediatedprovasculogenic eï¬ects are also observed on late epc he showed that ppar betadelta activation withgw501516 induced epc proliferation and tube formationwhereas epc treated with an inhibitor of cyclooxygenasecox or pgi2 synthase or with ppar betadeltaspeciï¬csirna also displayed an opposite eï¬ect furthermoreit has been demonstrated that ppar betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase mmp 9mediated insulinlikegrowth factor1 paracrine networks upon epc activation han also observed that ppar betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound overallin addition to ec ppar betadelta is also a key regulator ofepc or even may act as an initiator of activation of epc tofurther induce vasculogenesis ppar betadelta and tumor angiogenesislinked to tumor microenvironmentppar betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers []such as colonwhich suggests a crucial role of ppar betadelta in cancercells even though there exist some conï¬icting studies indicating that the functional role of ppar betadelta in tumorigenesis or carcinogenesis still remains highly controversial [] and dependent on speciï¬c tumor or cancer cell typesthus here we discuss the promotion of ppar betadelta intumor progression through facilitating tumor angiogenesisppar betadelta has been suggested as a critical hubnode transcriptional factor which governs a tumor angiogenic switch [ ] in the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in ppar betadeltanullmice in comparison with wildtype mice moreoverthe elevated ppar betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer indicating the crucial association of ppar betadelta withtumor angiogenesis progression and cancer invasivenessppar betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment tme where tumor angiogenesis is fostered moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withtme to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression for instance it wasshown that colon cancer cells with ppar betadelta knockoutfailed to stimulate ec vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that ppar betadelta is required for the promotion of angiogenesis in hypoxic stressmediated tme moreover in the tme tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites interestingly it hasbeen demonstrated that ppar betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an interleukin il10 dependent manner moreover impairedtumor growth and angiogenesis were observed in pparbetadelta ko bmt mice due to ppar betadelta deï¬ciencyin tumor myeloid cells suggesting that ppar betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of tmefurthermore the endoplasmic reticulum er an essential anelle involved in many cellular functions is implicated in tme in cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt er function and lead toaccumulation of unfolded proteins in er a condition knownas er stress cells adapt to er stress by activating an integrated signal transduction pathway called the unfolded protein response upr upr represents a survival response bythe cells to restore er homeostasis and has both survivaland cell death eï¬ects the mechanisms that determine cellfate during er stress are not well understood for instanceshort exposure to er stress initially increases akt signalingbut longterm er stress suppresses akt signaling ppar betadelta activation has been shown to reduce endoplasmic reticulum er stressassociated ammation inskeletal muscle through an ampkdependent mechanism and to reduce ammation in response to chronic erstress in cardiac cells furthermore it has been nicelyshown that ppar betadelta can repress rasoncogeneinduced er stress to promote senescence in tumors thisis mediated through the decrease of pakt activity promoting cellular senescence through upregulation of p53 and p27expression it would be interesting to investigate thedirect eï¬ects of ppar betadelta on senescence of tumorendothelial cells in an in vivo setting we recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and ï¬brotic lesions if ppar betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon ppar betadelta activationobserved by us and others [ ]most recently zuo demonstrated that pparbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including vegf and interleukin il8 most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspeciï¬c ppar betadelta overexpression in vivo leads to 0cppar researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ecspeciï¬cppar betadelta action mechanism in tumor progressionindependent of some controversial observations of pparbetadelta in speciï¬c tumor or cancer cell types wagner also ï¬rstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] crosstalk between ppar betadelta and signalmolecules ppar betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as vegf pdgf and fgfproinvasive matrixdegrading enzymes such as mmp9proammatory mediators such as cox2 and cytokinesand chemokines such as il1 and cxcl8 even some ofwhich have been further identiï¬ed as ppar betadelta directtarget genes besides a leading role of ppar betadelta amongthe signal molecules ppar betadelta may function in tmelinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules interplay between ppar betadelta and ammatoryangiogenesis ammatory angiogenesis is a crucial processin tumor progression for instance the proammatorymediator cyclooxygenase2 cox2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of vegf and induction of mmps moreover selectiveinhibition of cox2 has also been shown to suppress angiogenesis in vivo and in vitro it is well known that vegfaplays a critical role in both angiogenesis and vasculogenesis and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] ithas also been demonstrated that mmp9 triggers the angiogenic switch during carcinogenesis and enhances the availability of vegf to its receptors furthermore it hasbeen reported that ammatory cell mmp9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingmmp9 leptin is shown to mediate angiogenesisin vivo and in vitro through induction of ec proliferationand expression of mmp2 and mmp9 and to furtherpromote ec diï¬erentiation and directional migrationthrough enhancement of cox2 activity leptin couldalso induce angiogenesis via transactivation of vegfr inecs additionally besides inducing angiogenesisppar betadelta also functions in chronic ammationfacilitating tumorigenesis through induction of cox2 andits product prostaglandin e2 pge2 in vivo [ ]interestingly cox2 vegf mmp9 and leptin have beenidentiï¬ed as ppar betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells colorectal cancer cells [ ] epcs[ ] and liposarcoma cells respectivelylinks between vegf and mmpsin tme tumorltrating ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive mmp9and ammatory chemokines [] chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ecs towards tumor cells to form newvessels or indirectly by mediating immune ammatorycells to ltrate eventually promoting tumor angiogenesis chemotaxis of tumor cells and stromal cells in tmeis also required for tumor dissemination during tumor progression and metastasis [ ]cxc chemokines such as cxcl8 encoding il8 andcxcl5 are also involved in cox2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] it is further shown that il8 directly regulatesangiogenesis via recruitment of neutrophils whichfurther drives vegf activation moreoveril8responding neutrophils are considered as the major sourceof angiogenesisinducing mmp9 [ ] chemokine cc motif ligand ccl2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis furthermore myeloid monocytic cellssuch asmyeloidderivedtumormdscsassociated macrophages tams and dendritic cells arerecruited to the tumor site mainly by ccl2 and producemany proangiogenic factorssuch as vegf cxcl8plateletderived growth factor pdgf and transforminggrowth factor beta tgf beta [] in fact bothtgf beta and hypoxia are potentinducers of vegfexpression in tumor cells and collaborate with tme toprovide the foundation of tumor angiogenesis and cancercell invasion importantly il8 has been reported asa key target gene of ppar betadelta to promote angiogenesis in vivo and in vitro and ccl2 expression isalso signiï¬cantly upregulated upon vascular ppar betadelta overexpression in vivo suppressorcellscox2 also mediates il1 betainduced angiogenesisin vitro and in vivo [ ] il1 beta supports neovascularization through the regulation of the expression of vegfand its receptor vegfr2 flk1kdr on ecs il1 actsas an upstream proammatory mediator that initiates anddisseminates the ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ecs and leukocytes which facilitates tumorassociated angiogenesis in tme ammatory il1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as vegf vegffurther activates ecs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis in addition il6 also stimulates angiogenesis and vasculogenesis[ ] however gopinathan observed an il6induced newly forming vascular structure with defectivepericyte pc coverage ex vivo thus facilitating cancercell ltration and tumor metastasis through vascular leakage interestingly il1 and il6 expression levels are signiï¬cantly upregulated in the ppar betadelta overexpressionmouse model reported recently in summary ppar betadelta seems to act as a key leaderin ammatory mediatordriven tumor angiogenesis linkedto tme in which many proammatory mediators chemokines and proangiogenic factors closely communicate with 0cppar researcheach other and also associate with tumorltrating myeloid cells such as neutrophils tams and mdscs other key ppar betadeltamediated proangiogenicfactors it has been demonstrated that wilms tumor suppressor wt1 is a major regulator of tumor neovascularization andtumor progression e26 avian leukemia oncogene ets1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis in addition ets1 promotes cancer cell invasion throughupregulation of mmps consistent with this silencingof ets1 in highly invasive breast cancer cells also reducesthe expression of mmp9 and mmp1 ets1 also acts as a key regulator of mmps such asmmp1 mmp3 and mmp9 in human cancerassociatedï¬broblasts cafs [ ] cafs support tumor growthby secreting growth factors such as vegf fgf pdgf andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]cafs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ecmstructure thus helping a tumor to acquire an aggressive phenotype [ ] ppar betadelta in cafs also exhibits aprotumorigenic eï¬ect it was reported that ablation of pparbetadelta in cafs attenuated tumor growth by altering theredox balance in tme suggesting that ppar betadeltain cafs is also an important player in tumor developmentets1 induces the expression of vegf vegfr1 andvegfr2 in ecs [] in turn vegf is also a majorinducer of ets1 in ecs through the activation of either thepi3kakt pathway or the mekerk12 signal cascade[ ] wt1 is also reported to regulate tumor angiogenesis via direct transactivation of ets1 sryrelated hmgbox sox18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing and cancer progression and most recently it was further shown that speciï¬cecderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of sox18 interestingly theseimportant proangiogenic molecules including wt1 ets1and sox18 are also signiï¬cantly upregulated in the vascularppar betadelta overexpression model in vivo andwt1 is also identiï¬ed as a target gene of ppar betadeltain melanoma cells ppar betadelta may facilitate cancer progression atdiverse cellular levels in tme ppar betadelta activationis shown to induce colonic cancer stem cell csc expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the nanog gene nanog as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells and cancer cellsexpressing nanog also often exhibit stem cell properties protooncogene ckitcd117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in cscs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors moreover ckit has been identiï¬ed as a potentialmarker of the cancer stemlike cells in addition ckitnot only functions on ecs [ ] but also belongs to thetumor angiogenesispromoting molecule [] studiesalso suggested that activation of ckit enhances the expression of vegf that can be suppressed by imatinib an inhibitor of ckit in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] ckit is also involved in pathological ocular neovascularization and is regulated transcriptionally by wt1 and ppar betadelta pdgfb and its receptor pdgfr beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ecs [] andepcs and to regulate vascular permeability and vesselmaturation through recruitment of pericytes pcs and smooth muscle cells smcs in newly formingvessels moreover pdgfb and pdgfr beta also interactwith other proangiogenic factors such as fgf2 [ ]vegfa and its receptor vegfr2 furthermorepdgfb and pdgfr beta may also aï¬ect cancer growthand progression by directly acting on tme besides thecrosstalk with cafs [] pdgfr beta in stromalï¬broblasts may mediate pdgfbinduced tam recruitment thus implicating a role of pdgfr beta in tumorstroma to facilitate tumor progression most recently it wasfurther shown that speciï¬c targeting of pdgfr beta kinaseactivity in tme inhibited cancer growth and vascularizationin cancers with high pdgfb expression such as llc therefore this indicates the diverse role of pdgfb andpdgfr beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in tme pdgfr beta is demonstrated as a target of telomeric repeat binding factor trf2 that is further activated transcriptionally by wt1 pdgfb and pdgfr beta have further been identiï¬edas critical targets of ppar betadelta via a direct transactivation mechanism in vivo in conclusion a variety of key signal molecules involvedin tumor angiogenesis and tumor progression and metastasishave either been identiï¬ed as ppar betadelta direct targetsor largely upregulated in the vascular ppar betadelta overexpression model in vivo reported recently thus pparbetadelta activation seems to give rise to a highly angiogenicphenotype and even plays a hallmark role in promotingtumor angiogenesis and progression interestingly it appearsthat there could also exist a widely interactive networkbetween the downstream protumorangiogenic moleculesas described above therefore the crosstalk network is established between ppar betadelta and the various signal molecules and also between those molecules figure 1amoreover in addition to cancer cells ppar betadeltamay also produce pleiotropic eï¬ects in tme by modulatingdownstream key molecules to act on ecs epcs pcs smcscscs cafs and tumorltrating ammatory cells indirectly facilitating tumor angiogenesis and further promotingcancer development figure 1b other ppar betadelta target genes ppar betadeltaregulates the transcription oftarget genes via a direct 0cppar researchil1 betacox2leptinppar betadeltavegfvegfrpdgfr betatrf2wt1ets1mmp9pdgfbckitcxcl8il8il10ccl2cxcl8il8pdgfr betaappar betadeltaets1mmp9pdgfr betananogckittumorassociatedmacrophage tamdendritic cellneutrophilmyeloidderivedsuppressor cell mdscvegfmmp9pdgfbckitsox18pdgfr betaendothelial cell ece | 0 |
" discrete and compact nodular opacity (arrowheads) (B) focal neutrophil infiltration necrosis and hemorrhage (arrowheads) (H&E 12.5) (C) scattered small nodular opacities of lipiodol (long arrows) and faint nodular opacity (arrowheads) (D) focal hemorrhage and necrosis (arrowheads) with diffuse neutrophil infiltration (short arrows) (H&E 12.5). MLM in Group A (E F); (E) faint nodular lipiodol opacity (arrows) (F) focal hemorrhage (arrows) with diffuse neutrophil infiltration (arrowheads) (H&E 12.5). Methylene blue in Group A (G H); (G) faint nodular opacity (arrowheads) and (H) focal extent of neutrophil infiltration necrosis and hemorrhage (arrowheads) (H&E 12.5). Staining extent and localization ability of MLM versus methylene blue Data are mean±standard deviation. Numbers in parentheses are ranges. N/A indicates not available. *Non-parametric Mann-Whitney test was performed to compare the average score of MLM and methylene blue. MLM mixture of lipiodol and methylene blue. Localization ability score of staining and radio-opacity for MLM as well as methylene blue Data are numbers of subjects. Numbers in parentheses are percentages. MLM mixture of lipiodol and methylene blue. Comparison of localization ability between MLM and methylene blue in total subjects (n = 42) We considered a score of 2 or 3 as appropriate and 3 as excellent for localization respectively. Numbers in parentheses are percentages. *Fisher's exact test compared the proportion of appropriate or excellent staining between the mixture and methylene blue. MLM mixture of lipiodol and methylene blue. Localization ability of MLM: Evaluation of radio-opacity and staining score Data are given as numbers of subjects. Numbers in parentheses are percentages. MLM mixture of lipiodol and methylene blue. Histopathologic findings of lung specimens after percutaneous injections Data are numbers of subjects. Numbers in parentheses are percentages. N/A indicates not available. *Linear by linear association was performed between material and the extent of the histopathologic findings. Linear by linear association was performed between groups and the extent of the histopathologic findings. MLM mixture of lipiodol and methylene blue. PLoS One one 1932-6203 Public Library of Science San Francisco USA 24819391 4018408 PONE-D-13-46027 .0096911 Research Biology and Life Sciences Biochemistry Biomarkers Genetics Heredity Medicine and Health Sciences Diagnostic Medicine Epidemiology Biomarker Epidemiology Cancer Epidemiology Health Care Environmental Health Oncology Cancer Risk Factors Environmental Causes of Cancer Pathology and Laboratory Medicine Public and Occupational Health Pulmonology Environmental and Occupational Lung Diseases Single Nucleotide Polymorphism in ATM Gene Cooking Oil Fumes and Lung Adenocarcinoma Susceptibility in Chinese Female Non-Smokers: A Case-Control Study ATM Polymorphism and Risk of Lung Adenocarcinoma Shen Li 1 2 Yin Zhihua 1 2 Wu Wei 1 2 Ren Yangwu 1 2 Li Xuelian 1 2 Zhou Baosen 1 2 * 1 Department of Epidemiology School of Public Health China Medical University Heping District Shenyang Liaoning Province China 2 Key Laboratory of Cancer Etiology and Intervention University of Liaoning Province China Chang Jeffrey S. Editor National Health Research Institutes Taiwan * E-mail: bszhoumail.cmu.edu.cn Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: LS. Performed the experiments: LS YR XL. Analyzed the data: LS WW ZY. Contributed reagents/materials/analysis tools: LS ZY XL BZ. Wrote the paper: LS. Obtained informed consent from subjects: Baosen Zhou. 2014 12 5 2014 9 5 e96911 3 11 2013 12 4 2014 2014 Shen et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Background The ataxia-telangiectasia mutated (ATM) gene plays an important role in the DNA double-strand breaks repair pathway. Single nucleotide polymorphisms (SNPs) of DNA repair genes are suspected to influence the risk of lung cancer. This study aimed to investigate the association between the ATM -111G>A (rs189037) polymorphism environmental risk factors and the risk of lung adenocarcinoma in Chinese female non-smokers. Methods A hospital-based case-control study of 487 lung cancer patients and 516 matched cancer-free controls was conducted. Information concerning demographic and environmental risk factors was obtained for each case and control by a trained interviewer. After informed consent was obtained 10 ml venous blood was collected from each subject for biomarker testing. Single nucleotide polymorphism was determined by using TaqMan method. Results This study showed that the individuals with ATM rs189037 AA genotype were at an increased risk for lung adenocarcinoma compared with those carrying the GA or GG genotype (adjusted odds ratios (OR) 1.44 95% confidence interval (CI) 1.022.02 P?=?0.039). The stratified analysis suggested that increased risk associated with ATM rs189037 AA genotype in individuals who never or seldom were exposed to cooking oil fumes (adjusted OR 1.89 95%CI 1.033.49 P?=?0.040). Conclusions ATM rs189037 might be associated with the risk of lung adenocarcinoma in Chinese non-smoking females. Furthermore ATM rs189037 AA genotype might be a risk factor of lung adenocarcinoma among female non-smokers without cooking oil fume exposure. This study was supported by grant no. 81272293 from National Natural Science Foundation of China grant no. 81102194 from National Natural Science Foundation of China and grant no. 00726 from China Medical Board. The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Lung cancer is the leading cause of cancer-related deaths both worldwide and in China. Although cigarette smoke is the major risk factor for lung cancer only a fraction of smokers develop this disease [1] suggesting that host genetic susceptibility may play an important role in the development of lung cancer. Recent genetic susceptibility studies of lung cancer have focused on single nucleotide polymorphisms (SNPs) in candidate genes among which DNA repair genes are increasingly studied because of their critical role in maintain genome integrity. Genetic variations in DNA repair genes are thought to affect DNA repair capacity and deficits in DNA repair capacity may lead to genetic instability and carcinogenesis [2] [3]. As one of the DNA repair genes ataxia-telangiectasia mutated (ATM) gene which is responsible for the multisystem autoxomal recessive disorder ataxia-telangiectasia (AT) plays a crucial role in the recognition signaling and repair of DNA damage especially DNA double-strand breaks (DSBs) [4] [5]. The ATM protein is a member of phosphoinositide 3-kinase (PI-3 kinases) and can be activated by DSBs caused by ionizing radiation or reactive oxygen intermediates [6] [7]. Once activated ATM can phosphorylate various downstream substates that function in cell cycle arrest apoptosis and DNA repair such as p53 NBS1 BRCA1 and Chk2 [8] [9]. Therefore genetic variants in ATM gene may lead to the structure and function change of the protein and act as important factors indicating individual susceptibility to cancer. ATM -111G>A (rs189037) resides in the promoter of ATM gene. Increasing studies have shown that variations in the DNA promoter sequence may potentially alter the affinities of multiple regulatory proteins-DNA interactions or the specificity of the transcriptional process [10][13]. Although this polymorphism makes no amino acid change the alleles may have different binding affinity to the transcription factor and exhibit different levels of mRNA expression [14] [15]. Zhang et al. [16]declared that ATM rs189037 AA genotype was associated with a lower ATM mRNA levels than GG genotype in lung tissue samples. Their results showed that the G-to-A change might create a transcriptional inhibitor-binding site for ATM rs189037 A allele promoter and subsequently reduce the ATM mRNA expression. Consequently lower expression of ATM might cause elevated sensitivity to ionizing radiation defects in the activation of cell cycle checkpoints a reduced capacity for DNA repair and abnormal apoptosis. All of these features would contribute to increased individual cancer susceptibility. In recent years a number of studies have evaluated the association between this polymorphism and cancer risk such as thyroid carcinoma [17] oral cancer [18] breast cancer [19] leukemia [20] nasopharyngeal carcinoma [21] glioma [22] and lung caner [23][25]. Previous studies of ATM rs189037 have included cigarette smokers as cases and controls that made it difficult to judge whether this polymorphism were associated with lung cancer or tobacco use. Considering the facts in China the incidence and death rate of lung cancer in women continues to increase and this phenomenon is frequently occurring in those who have never smoked. In order to have a better control of confounding of gender or smoking we performed a case-control study to identify the association between the polymorphism of ATM rs189037 and the risk of lung cancer in the non-smoking females in Chinese Han population. We also investigated the interaction between genetic polymorphism and environmental exposure in lung cancer. Methods Subjects This hospital-based case-control study included 487 lung cancer patients and 516 cancer-free hospital controls. All subjects were female non-smokers and they were from unrelated ethic Han Chinese. The cases were recruited during January 2002 to November 2012 at Liaoning Cancer Hospital & Institute. All patients were histologically confirmed to have lung cancer before any radiotherapy and chemotherapy. During the same time controls were selected from patients with other lung diseases but free of cancer history and symptom. Controls suffered mainly from bronchitis pneumonias fibrosis sarcoidosis chronic obstructive pulmonary disease and emphysema. Controls were all non-smoking females and frequency-matched to case subjects for age (±5 years). This study was approved by the institutional review board of China Medical University and written informed consent was obtained from each participant or each participant's representatives if direct consent could not be obtained. Data Collection A total of 10 ml of venous blood was collected from each patient. Patients were interviewed to collect information for demographics and environmental exposure at the time they were admitted to hospital. Information concerning demographic characteristics passive smoking cooking oil fume exposure fuel smoke exposure family history of cancer occupational exposure and dietary habit was obtained for each case and control by trained interviewers. An individual was defined as a smoker if she had consumed a total of 100 cigarettes in her lifetime; otherwise she was considered as a non-smoker. About fuel smoke exposure participants who used coal-fuel-burning stoves without chimneys were regarded as fuel smoke exposure. For exposure to cooking oil fumes participants were mainly asked about the method of cooking and eyes or throat irritation. For cooking methods participants were asked whether they cooked food in a stir-frying way and how many times a week; for eyes or throat irritation participants were asked how often they felt eyes or throat irritated by the oily smoke. There were four possible responses ranging from never seldom sometimes and frequently. Subjects were considered as cooking oil fume exposure if they met criteria as follows: (1) have cooked for over 15 years; (2) cooked food in a stir-frying way for more than twice a week; (3) felt eyes or throat irritated by oily smoke. Exposure for cooking oil fume was categorized as an indicator variable equal to 1 if participants reported frequently or sometimes and equal to 0 otherwise. Genotype Analysis Genomic DNA was extracted from peripheral blood samples by the conventional phenol-chloroform extraction method. SNP was genotyped by investigators blinded to case-control status in order to avoid any genotyping bias using TaqMan methodology and read with the Sequence Detection Software on an Applied Biosystems 7500 FAST Real-Time PCR System according to the manufacturer's instructions (Applied Biosystems Foster City CA). Amplification was done under the following conditions: 95°C for 10 min followed by 47 cycles of 92°C for 30 s and 60°C for 1 min. In this study 487 lung cancer patients and 516 controls were all genotyped successfully and 5% duplicated samples were randomly selected to assess the reproducibility for quality control with a concordance rate of 100%. Statistical Analysis The x2 test and t test were applied to estimate differences in demographic variables and distributions of genotypes between cases and controls. The association of genotypes of ATM rs189037 with risk of lung cancer was estimated by computing the odds ratios (ORs) and 95% confidence intervals (CIs) in unconditional logistic regression analysis. The Hardy-Weinberg equilibrium (HWE) was tested using goodness-fit x2 test to compare the genotype frequencies in the control subjects from those expected. A logistic regression model was used to evaluate gene-environment interactions. All data were analyzed with Statistical Product and Service Solutions (SPSS) v13.0 for Windows if not otherwise specified. All statistical analysis were two-sided and the significance level was set at P<0.05. Results Population characteristics A total of 487 lung cancer and 516 age-matched cancer-free controls were enrolled in this study. As shown in the mean ages of cases and controls (mean ±S.D.) were almost identical (56.5±11.7 and 56.3±12.5 respectively). All cases were female non-smoking lung cancer patients. No statistically significant difference was found between cases and controls in terms of age (P?=?0.248) and monthly income (P?=?0.084). Cases included 434 non-small cell lung cancer (NSCLC) patients and 53 small cell carcinoma patients. In the NSCLC cases there were 320 adenocarcinomas 73 squamous cell carcinomas and 41 other tumors with a variety of different pathologies (such as large cell carcinomas mixed cell carcinomas or undifferentiated carcinomas). .0096911.t001 Characteristics of lung cancer cases and controls. Variables Cases(%) Controls(%) P value Female 487 516 Mean age (years) 56.5±11.7 56.3±12.5 0.248a Income (yuan/month) 628.9±419.3 563.5±387.6 0.084a Never smoker 487 516 Histological type NSCLC 434(89.1) Adenocarcinoma 320(65.7) Squamous cell carcinoma 73(15.0) Small cell carcinoma 53(10.9) Other 41(8.4) a Student's t-test was used to compare the frequency distributions of demographic variables between the cases and controls. Association analysis The observed genotype frequencies among the control subjects was in agreement with that expected under the Hardy-Weinberg equilibrium (P?=?0.119). The distribution of ATM rs189037 genotypes among subjects were displayed in . " | 1 |
"Type and diabetes confer an increased risk of pancreatic cancer PaC of similar magnitudesuggesting a common mechanism The recent finding that PaC incidence increases linearly with increasing fastingglucose levels supports a central role for hyperglycaemia which is known to cause carbonyl stress and advancedglycation endproduct AGE accumulation through increased glycolytic activity and nonenzymatic reactions Thisstudy investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanismsinvolvedMethods Pdx1CreLSLKrasG12D mice were interbred with mitosis luciferase reporter mice rendered diabetic withstreptozotocin and treated or not with carnosinol FL92616 a selective scavenger of reactive carbonyl speciesRCS and as such an inhibitor of AGE formation Mice were monitored for tumour development by in vivobioluminescence imaging At the end of the study pancreatic tissue was collected for histologyimmunohistochemistry and molecular analyses Mechanistic studies were performed in pancreatic ductaladenocarcinoma cell lines challenged with high glucose glycolysis and glycoxidationderived RCS their proteinadducts AGEs and sera from diabetic patientsResults Cumulative incidence of invasive PaC at weeks of age was in untreated diabetic vs in FL92616gtreated diabetic and in nondiabetic mice FL92616 treatment suppressed systemic and pancreaticcarbonyl stress extracellular signalregulated kinases ERK activation and nuclear translocation of Yesassociatedprotein YAP in pancreas In vitro RCS scavenging and AGE elimination completely inhibited cell proliferationstimulated by high glucose and YAP proved essential in mediating the effects of both glucosederived RCS andtheir protein adducts AGEs However RCS and AGEs induced YAP activity through distinct pathways causingreduction of Large Tumour Suppressor Kinase and activation of the Epidermal Growth Factor ReceptorERKsignalling pathway respectivelyContinued on next page Correspondence giuseppepuglieseuniroma1it Stefano Menini and Carla Iacobini contributed equally to this work1Department of Clinical and Molecular Medicine La Sapienza University Viadi Grottarossa Rome ItalyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMenini Journal of Experimental Clinical Cancer Research Page of Continued from previous pageConclusions An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINsprogression to invasive PaC showing that hyperglycaemia promotes PaC mainly through increased carbonyl stressIn vitro experiments demonstrated that both circulating RCSAGEs and tumour cellderived carbonyl stressgenerated by excess glucose metabolism induce proliferation by YAP activation hence providing a molecularmechanism underlying the link between diabetes and PaC and cancer in generalKeywords Pancreatic ductal adenocarcinoma Hyperglycaemia Reactive carbonyl species Methylglyoxal Advancedglycation endproducts Carnosine derivatives Yesassociated protein Large tumour suppressor kinase Epidermalgrowth factor receptor Extracellular signalregulated kinases BackgroundPancreatic cancer PaC is the tenth most common incident cancer but the seventh leading cause of cancerrelated death worldwide [] because of the poor 5yearsurvival outcomes [] Due to the rising prevalence ofrisk factors such as obesity and type diabetes PaC isexpected to become the second leading cause of cancerrelated death in the US by [] Type diabetes wasfound to be associated with a 7fold higher risk ofPaC in the first year after diabetes diagnosis and nearlytwofold thereafter [ ] Though type diabetes is themain contributor to this problem the entity and temporal trajectory of PaC risk were recently reported to besimilar in type diabetes [] suggesting a commonmechanism related to hyperglycaemia This concept issupported by the recent finding that PaC incidence increases linearly with increasing fasting glucose levelseven within the normal range []thus hindering the understanding ofPrevious studies have shown that type diabetes induced by a highfat diet promotes PaC [ ] Howeverthis experimental model of the metabolic syndrome doesnot allow assessing the role of hyperglycaemia independent of confounding factors such as obesity and hyperinsulinemiathemechanisms underlying the risk conferred by hyperglycaemia We have recently demonstrated that advancedglycation endproducts AGEs promote proliferation ofhuman pancreatic ductal adenocarcinoma PDA cell linesand that exogenous AGE administration markedly accelerates invasive tumour development in a mouse model ofKrasdriven PaC [] Accumulation of AGEs in diabetesis mainly due to increased formation of reactive carbonylspecies RCS derived from glucose autooxidation egglyoxal GO but also from cell metabolism of excess glucose through glycolysis eg methylglyoxal MGO []In turn RCS react with amino groups of proteins causingstructural and functional modifications The resulting irreversible adducts ie AGEs accumulate in tissues wherethey can exert further biological effects through interaction with specific receptors [ ]Carnosine betaalanylLhistidine is an endogenousinhibits AGEhistidinecontainingdipeptidethat[]carnosine derivativesformation by scavenging RCS [] Though LCarnosinewas proven to be effective in several carbonyl stressrelated disease conditionsincluding metabolicdisorders []its therapeutic use in humans ishampered by the presence of high levels of serum carnothus prompting the search for carnosinasesinase[] The novelresistantbioavailable compound carnosinol ie 2S23aminopropanoylamino31Himidazol5ylFL [] was shown to be highly effective in attenuating diabetesassociated vascular complications [] and obesityrelated metabolic dysfunctions [ ]Moreover it was recently shown that Lcarnosine is effective in counteracting glycolysisdependenttumourgrowth by quenching RCS []propanollesionsthe progression of preneoplasticThis study aimed at investigating whether hyperglycaemia associated with experimental type diabetes favourstomalignancy in a wellvalidated mouse model of PaC byincreasing carbonyl stress To this end mice weretreated with the RCS scavenger and inhibitor of AGEformation FL92616 An additional objective was toanalyse the effect of the diabetic milieu and of FL926 on the activity of Yesassociated protein YAP a keydownstream target of KRAS signalling required for progression of pancreatic intraepithelial neoplasias PanINsto invasive PaC [ ] and for MGOinduced tumourgrowth []MethodsIn vivo studyThe experimental protocols comply with the principles ofwwwnc3rsukarriveguidelines and were approved by the National Ethics Committee for Animal Experimentation ofof HealthAuthorization no 14702015PR The mice were housedin single cages with woodderived bedding material in aspecific pathogenfree facility with a 12h lightdark cycleunder controlled temperatures °C Mice werecared for in accordance with the Principles of LaboratoryAnimal Care National Institutes of Health publ no revised and with national laws and receivedItalian Ministrythe 0cMenini Journal of Experimental Clinical Cancer Research Page of water and food ad libitum The primary and secondaryendpoint were the development of invasive PaC and thedevelopmentprogression of PanINs respectivelyDesignThe effect of diabetes on PaC progression was investigatedin Pdx1CreLSLKrasG12D KC mice which develop autochthonous PaC in a pattern recapitulating human pathology with high fidelity by developing the full spectrum ofPaC progression from preneoplastic lesions PanINs toadenocarcinoma and metastasis [ ] KC mice wereinterbred with mitosis luciferase MITOLuc reporter miceto obtain KCMito KCM mice [ ] The LSLKrasG12D lineage was maintained in the heterozygousstate Mice were screened by polymerase chain reactionPCR using tail DNA amplified by specific primers to theLoxP cassette flanking mutated KrasG12D wild type KrasCre recombinase and MITO genes as previously reported[ ] In the MITOLuc mouse an artificial minimal promoter derived from the cyclin B2 gene and induced by NFY drives the expression of the luciferase reporter specificallyin replicating cells Therefore both normal eg bone marrow and tumour actively proliferating cells may be localized by a bioluminescence imaging BLIbased screen [ ] We have previously shown that KCM mice developpreinvasive PanINs and invasive ductal PaC with thesame penetrance latency and histological features as thosedescribed for KC mice [] According to the Ethics Committee recommendations to limit the number of animalsthe experiments were stopped when it was sufficient toconfirm or reject the working hypothesis in a statisticallyand clinically meaningful mannerFigure shows the flowchart and timeline of study design Thirtythree KCM mice were rendered diabeticFig Flowchart and timeline of study design Please refer to the text for detailed description In dashed boxes groups of nondiabetic KCMmice Ctr that served as control for the effect of STZ STZnonDiab or FL92616 FL treatment Ctr FL on PaC development and progressionTo avoid unnecessary suffering three diabetic mice Diab and one Diab mouse treated with FL DiabFL were killed and weeks respectivelybefore the end of the study STZ streptozotrocin BLI bioluminescence imaging 0cMenini Journal of Experimental Clinical Cancer Research Page of with streptozotocin STZ and followed for weeksie up to weeks of age After an overnight fast weekold mice were intraperitoneally injected with mg·kg STZ SigmaAldrich St Louis MO USA Success rate defined as the percentage of STZinjected micewith glucose levels mgdL for the entire studyperiod was Three days after injection diabetic mice were randomized to receive no treatmentDiab n or FL92616 gift of Flamma SpAChignolo dIsola Italy [] at a dose of mg·kg day in the drinking water DiabFL n andinjected weekly with IU of insulin glargine to preventexcessive weight loss and ketoacidosis FL92616 wasshown to have a suitable absorption distribution metabolism excretion and toxicity ADMET profile and thegreatest potency and selectivity toward RCS among allother carnosine derivatives [] The FL92616 dosewas chosen based on previous results from our group[]showing high efficacy in preventing diabetesinduced renal injury and from other investigators indicating a good safety profile at the dose of g·kg ·day [ ] Neither histological abnormalities of theliver kidney lung and heart nor functional abnormalities attributable to toxicity on these tissues wereobserved in this study or in a previous one [] STZtreated mice not fulfilling the criteria for diabetes diagnosis STZnonDiab n served as control for STZeffect on PaC seven of these mice failed to develophyperglycaemia whereas two had spontaneous recoveryfrom diabetes within weeks Vehicle salineinjectedKCM mice were used as nondiabetic controls and either left untreated Ctr n or treated with FL926 Ctr FL n to check for any drug effectMice were subjected to in vivo BLI every otherweek [ ] and daily manual palpation of the abdomen to check for tumour growth and avoid the lossof animals along with the need to cope with the related ethicalissues ie compliance with the 3Rsprinciples Briefly min after administration of Dluciferin mgkg body weightintraperitoneal Perkin Elmer Hopkinton MA USA photon emissionfrom the different body areas was acquired for minand analysed with a CCD camera Xenogen IVIS Lumina System Perkin Elmer A specific region ofinterest ROI corresponding to the abdominal areaoccupied by the pancreas was manually selected andthe total photon flux ps from this ROI was evaluated with Living Image software Caliper Life Sciences Perkin Elmer [ ]At the end of the study mice were anaesthetizedwith ketamine mg·kg Imalgene ip and xylazine mg·kg Rompum ip and killed by cervical dislocation According to the Ethics Committeerecommendations to avoid suffering three Diab andone DiabFL mice presenting with both positive BLIand a palpable abdominal mass or poor general condition were killed and weeks respectively beforethe end of the study The lungs and the middle partof the gastrointestinal tractincluding the pancreasand the liver were dissected and exposed to theCCD camera for min for photon emission assessment The pancreas was dissected photographed andthen one part was stored at °C forweightedmolecular analysis whereas the other part was processed for histologicalimmunohistochemical analysis[] At time of collection a technician CC seeAcknowledgementstoallow blinded analysisrecoded biologicalsamplesMetabolic parametersBody weight and blood glucose were monitored weeklyAt the end of the study the levels of haemoglobin HbA1c an indicator of longterm glycaemic control wereassessed by using the Mouse HbA1c Assay Kit Crystal Chem Zaandam Netherlands and serum AGEsand total protein carbonyls PCOs two carbonyl stressmarkers were measured by ELISA OxiSelect¢ Advanced Glycation EndProduct Competitive ELISA Kitno STA817 and OxiSelect¢ Protein Carbonyl ELISAKit no STA310 respectively Cell Biolabs Inc SanDiego CA USAPancreas histologySix 4μmthick nonserial pancreatic sections stainedwith haematoxylin and eosin were examined to confirm the presence of invasive PaC Pancreas withoutinvasive PaC were analysed to grade dysplastic ductsie PanINs according to previously established criteria [] The numbers oflowgrade PanIN1ABand highgrade PanIN23 dysplastic ducts werecounted and expressed as a percentage of total ductsin the specimen []Pancreatic AGEsERK phosphrylation status nuclear YAP and itstarget gene connective tissue growth factor CTGFLevels of AGEs pERK and CTGF protein in homogenates and of active nonphosphorylated YAP1 innuclear extracts of pancreas of mice were assessed byWestern blot Human PDA tissues n were obtained from the Pathology Unit of SantAndrea HospitalRome Italyin agreement with the ethical guidelinesestablished by the locally appointed Ethics CommitteePancreatic tissue distribution of AGEs and activatedYAP1 in mouse and human specimens were evaluatedby dual label immunofluorescence and immunoperoxidase respectively [ ] For immunofluorescence agoat polyclonalrabbitantiAGE antibodyand a 0cMenini Journal of Experimental Clinical Cancer Research Page of sections weremonoclonal antibody to active nonphosphorylatedYAP1 were used as primary antibodies followed by appropriate secondary fluorescent antibodies see Supplementary Table S1 for antibodies in Additional file Sections were analysed at a fluorescence microscopeZeiss AXIO A1 equipped with an Axiocam colorcamera Carl Zeiss Italy Milan Italy For immunoperoxidase formalinfixed paraffin embedded sections μm thick were rehydrated and treated with H2O2in PBS for min to block endogenous peroxidase activity Heat mediated antigen retrieval was performed withAntigen Unmasking Solution Citric Acid Based H Vector Laboratories Burlingame CA USA forAGE staining or TrisEDTA buffer pH for YAPstaining both for min Nonspecific binding wasblocked by incubation in Protein block serum free AgilentDako Santa Clara CA USA for min at roomtemperature Thenincubated withAvidinBiotin blocking Kit SP2002 Vector Laboratories for min an antiAGE antibody Abcam Cambridge UK ab23722 or an antibody directed to theactive nonphosphorylated YAP1 Abcam ab205270at °C overnight and the appropriate biotinylated secondary antibody at room temperature for min seeSupplementary Table S1 for antibodies in Additional file Finally sections were stained with UltraTek Horseradish Peroxidase ABL015 ScyTek Laboratories UTUSAfor min followed by 33diaminobenzidineDABH2O2 ChromogenSubstrate Kit High ContrastACV500 ScyTek Laboratories until the reaction product was visualized min and counterstained withhematoxylin AGE positive staining and nuclear expression of YAP were measured in random fields of eachsection at a final magnification of 250X and 400X respectively by means of the interactive image analyzerImagePro Premier ImmaginiComputer MilanItaly AGE positivity was expressed as the mean percentage of fields area occupied by the specific stain Expression status of active YAP in tumor specimens wasassessed by counting the number of nuclei positive forYAP and expressed as the mean ratio of YAPpositive nuclei to total nucleiand AGEstheIn vitro studyThe in vitro study investigated the putative role ofRCStumourpromoting effect of high glucose HG and the protective effect of the carbonylsequestering agent andAGE inhibitor FL92616as mediators ofDesignHuman MIA PaCa2 Catalogue No Lot No14A02 and Panc1 Catalogue No Lot No10G011 cells SigmaAldrich were used for assessing theeffects of HG and FL92616 on cell proliferation Experiments aimed at investigating the molecular mechanismsunderlying the glucosemediated effects and the protection by FL92616 were conducted on MIA PaCa2 cellsMycoplasma contamination in cell cultures was regularlytested by PCR MycoSPY Kit Biontex Laboratories GmbHMunchen Germany Human PDA cells were maintainedin DMEM supplemented with FBS and incubated indifferent conditions for three daysie normoglycaemia normal glucose mM hyperglycaemia HG mM treated with MGO or GO μM SigmaAldrich two RCS and AGE precursors or the preformed AGE Nεcarboxymethyllysine CML μgmLprepared as previously reported [ ] with or withoutFL92616 mM and exposed to DMEM lowglucose medium containing of pooled sera from nondiabetic or diabetic individuals before and after AGE removalfrom diabetic serum by an immunoadsorptionmethod see below with or without FL92616 mMInformed consent was obtained from nondiabetic anddiabetic individuals Moreover both YAP and EpidermalGrowth Factor Receptor EGFR were silenced to assessthe role of YAP and EGFR pathway in RCS and AGEinduced cell proliferation see belowRemoval of AGEs from diabetic serumAGEs were removed from diabetic serum using animmunoadsorption method To immunoprecipitateAGEmodified proteins μl of diabetic serum wasincubated for h with μl of Pierce NHSactivatedmagnetic beads Thermofisher Scientific covalentlyconjugated with μg of antiAGE antibody Abcamsee Supplementary Table S1 for antibodies in Additional file according to the manufacturer instruction To confirm the efficiency of AGE depletionAGE concentration in both treated unbound serumfraction and untreated diabetic serum was evaluatedin triplicate by ELISA OxiSelect¢ Advanced GlycationEndProduct Competitive ELISA Kit no STA817Cell Biolabs Inc San Diego CA USA Followingthis procedure the concentration of AGEs in diabeticserum was reduced by about reaching a concentration similar to that of the nondiabetic serum seethe Results sectionYAP and EGFR silencingYAP and EGFR were silenced using smallinterferingRNAs siRNAs and irrelevant scrambled siRNAs as control Thermo Fisher Scientific Waltham MA USAValidated predesigned siRNA oligonucleotides and related TaqMan assays are detailed in SupplementaryTable S2 see Additional file Lipofectamine RNAiMAX Thermo Fisher Scientific transfections were performed using nM of each siRNA 0cMenini Journal of Experimental Clinical Cancer Research Page of Cell survival and proliferationCell viability and proliferation were evaluated by Cytoselect WST1 Cell Proliferation Assay Cell Biolabs following the manufacturer instructionsYAP1 its upstream regulators large tumour suppressor Kinase 1LATS1 and EGFRERK pathway and itsmolecular targets CTGF WTN5A and EMP2 in inhuman PDA cells Cells were extracted in SDS buffer containing protease and phosphatase inhibitorsSigma Aldrich Nuclear protein extracts were obtainedfrom cell monolayers with the Nuclear Extract Kit Active Motif Corp Carlsbad CA USA Protein concentrations were determined using the Bradford Assay KitBioRad Hercules CA USA Nuclear protein levels ofYAP1 and cellular protein levels of total and EGFRphosphorylated at Tyr1068 pEGFR total and pERK and LATS1 a key kinase of the Hippo pathway []were assessed by Western blotting see SupplementaryTable S1 for antibodies in Additional file KRAS activity was evaluated by the KRAS activation Assay Kit noSTA400K Cell Biolabs Inc according to the manufacturers protocol Briefly mg of lysate was subjected topulldown and μg of lysate was used to measure totalKRAS Pulldown and totallysates were subjected toWestern blotting procedure using the primary antibodyagainst KRAS provided by the kit The mRNA levels ofCTGFCCN2 WTN5A and EMP2 three recognized molecular targets of YAP [ ] were assessed by realtime PCR RTPCR using a StepOne RealTime PCRSystem and TaqMan Gene Expression assays ThermoFisher Scientific [] listed in Supplementary Table S3see Additional file Statistical analysisResults are expressed as mean ± SD mean ± SEM orpercentage Differences between cell typestreatmentsor animal groups were assessed by oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisons or twoway ANOVA followed bythe Bonferroni posttest as appropriate Betweengroup differencesin PaC incidence were assessedusing the Chisquared test and Fishers exact test tocompute a Pvalue from a contingency table A Pvalue of was considered to be significant Allstatisticalincluding linear regression analysiswere performed on raw data using GraphPad Prismversion for Windows GraphPad Software SanDiego CA USAtestsResultsIn vivo studyMetabolic parametersSTZtreated KCM mice developed hyperglycaemiastartingandabout h postinjection Fig 2ashowed a slight decline in the growth curve vs Ctrmice which reached statistical significance only at and weeks of age Fig 2b Despite no differencein body weight Fig 2c blood glucose Fig 2d andHbA1c levels Fig 2e FL92616 treatment preventedthe diabetesassociated increase in circulating AGEsFig 2f and total PCOs Fig 2g as assessed at theend of the studyInvasive PaC development Representative BLI imagesat the end of the study period and total photon fluxinduction from pancreas at and weeks of ageare shown in Fig 3a At sacrifice pancreas weightwas significantly P increased in Diab ± g vs Ctr ± g and vs DiabFL ± g KCM mice Pancreasbody weight percent ratiowas almost tripled in Diab vs Ctr mice whereas nostatistical difference was observed between DiabFLand Ctr mice Fig 3b and Table As assessed byhistology Fig 3c cumulative incidence ofinvasivePaC at weeks of age was in Diab mice vs in DiabFL and in Ctr mice Fig 3d and Table Representative BLI images and pancreas histologyfrom Ctr Diab and DiabFL are shown in Fig 3cdNeither the Ctr FL nor the STZnonDiab groupshowed significant differences in the incidence invasive PaC and pancreasbody weight percent ratio vsthe Ctr group Table Furthermore no betweengroup differences were observed in tumour invasiveness except for an apparent reduction in DiabFL vsDiab group Table However the few cases of PaCin DiabFL n and Ctr n mice prevent toperform statistical comparisons among groupsformetastatic disease Representative ex vivo BLI andhistology images of liver and lung metastases are presented in Supplementary Fig S1 in Additional file Grading of dysplastic ducts in mice free of invasivePaC Table showed significant differences betweenDiabFL and Diab mice for the percentage of normalducts which was higher and of highgrade PanINswhich was lowerin the FL92616 treated arm Inaddition Ctr FL mice presented with higher normalducts and lowerlowgrade PanINs vs Ctr micewhereas no difference was observed between STZnonDiab and Ctr micePancreatic AGEs ERK phosphrylation status nuclear YAP and connectivegrowth factorCTGF Pancreatic accumulation of AGEs Fig 4aand levels pERK Fig 4b CTGF Fig 4c awellestablished transcriptional target of YAP [ ] and nuclear YAP1 Fig 4d were increased inDiab vs Ctr mice and increments were prevented bytissue 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig Glucose and HbA1c levels body weight and hyperglycaemiaassociated carbonyl stress Blood glucose levels and body weight during thestudy period a and b and at the end of the study period weeks of age1 c and d and HbA1c levels e and serum levels of AGEs f andtotal PCOs g at the end of the study period weeks of age1 in control Ctr Ctr treated with FL92616 Ctr FL diabetic Diab and Diabtreated with FL92616 DiabFL KCM mice Statistical significance between groups for time course of blood glucose a and body weight c wascalculated using twoway ANOVA followed by the Bonferroni posttest Each time point represents mean ± SD of animals until the 17th weekof age and animals from the 18th to the 22nd week of age Statistical significance for blood glucose c body weight d serum levels ofAGEs e and PCOs f at weeks of age1 was assessed using oneway ANOVA followed by the StudentNewmanKeuls test for multiplecomparisons Each dot represents one case and bars represent mean ± SEM P or P vs Ctr P vs Diab 1Except for threeDiab and one Diab FL mice which were killed and weeks respectively before the end of the study see Results section for further detailslabelFL92616 treatment Dualimmunofluorescenceanalysis confirmed the association between AGEs andnuclear YAP1 in PaC lesions from Diab mice Fig4e A significant positive relationship between AGEaccumulation and nuclear YAP1 levels was also observed in human PDA Fig 4fgIn vitro studyProliferation of human PDA cellsHG concentration mimicking diabetic hyperglycaemiapromoted PDA cell growth and this effect was prevented by FL92616 Fig 5ab The AGE precursors RCS MGO and GO and the preformed AGE 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig In vivo BLI and gross and microscopic examination of pancreas Representative BLI at the end of the study period and total photon fluxps induction from pancreas at and weeks of age1 a pancreasbody weight percent ratio b representative pancreas histology coriginal magnification 100X scale bar μm and cumulative incidence of PaC d in control Ctr diabetic Diab and Diab treated with FL DiabFL KCM mice at the time of sacrifice Statistical significance between groups for pancreasbody weight percent ratio a wascalculated using oneway ANOVA followed by the StudentNewmanKeuls test for multiple comparisons Each dot represents one case and barsrepresent mean ± SEM Statistical significance for PaC incidence b was assessed using the Chisquared test and Fishers exact test P vsCtr P vs Diab Is islet invasive PaC arrows PanINs 1Except for three Diab and one Diab FL mice which were killed and weeksrespectively before the end of the study see Results section for further detailsCML also stimulated PDA cell proliferation FL926 was able to inhibit cell proliferation induced byMGO and GO but not CML Fig 5c Treatmentwith CML but not with MGO induced ERK activation and FL92616 was ineffective in counteractingphosphorylation status Fig 5d However the proliferating effect of both the RCS MGO and the AGECML was associated with YAP1 nuclear persistenceand activity Again FL92616 efficiently preventedeffectCMLtheofonERKthe nuclear translocation of YAP1 induced by MGObut failed to counteract the effect of CML Fig 5eConsistently FL92616 treatment reversed the MGOinduced upregulation of gene expression of CTGFWnt Family Member 5A WNT5A and EpithelialMembrane Protein EMP2three wellrecognizedYAP target genes [ ] Conversely FL92616was ineffective in preventing the modulatory effect ofCML on the mRNA level of these genes Supplementary Fig S2 in Additional file 0cMenini Journal of Experimental Clinical Cancer Research Page of CtrDiab1Diab FL1Ctr FL ± ± ± Table Pancreatic cancer PaC incidence PancreasBodyweight Wt percent ratio and metastasisPaC NtotPancreasBody Wt Metastasis Ntot PaC ± STZnonDiab ± Cumulative incidence of PaC and PancreasBody weight Wt percent ratio incontrol Ctr diabetic Diab Diab treated with FL92616 DiabFL Ctr treatedwith FL92616 Ctr FL and streptozotocintreated nondiabetic STZnonDiab KCM mice at the end of the study weeks of diabetes weeks ofage1 The number of KCM mice with metastasis liver and or lung on thetotal number of PaC cases is also shown KCM LSLKrasG12D Pdx1Cre MITONtot number of casestotal number of mice Ntot PaC number of casestotal number of PaC PaC ductal adenocarcinoma and hepatic andor lungmetastasis were confirmed by histology P or P vs Ctr P or P vs Diab Statistical significance between groups forPancreasBody Weight percent ratio was calculated using oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisonsStatistical significance for PaC rate was assessed using the Chisquared testand Fishers exact test Except for three Diab and one Diab FL mice which were killed and weeks respectively before the end of the studyMechanisms underlying RCS and AGEinduced YAPactivationSilencing of YAP1 using two independent siRNAssiYAP1 and Fig 6a significantly inhibitedthe transcription activity of YAP target genes induced by both MGO and CML in PDA cells Fig6b In MGOtreated cells YAP induction was associated with a decrease in protein levels of LATS1 awellestablished negative regulator of YAP activity[] whereas CML treatmentfailed to modulateLATS1 Fig 6c Instead treatment with CML butnot with MGO was found to induce EGFR phosphorylation pEGFR Fig 6d EGFR silencing Fig6e almost completely reversed YAP1 nuclear translocation Fig 6f KRAS activation and ERK phosphorylation Supplementary Fig S3ABinduced by CMLEffects of serum from diabetic patients on proliferation ofhuman PDA cellsThe levels of AGEs were ± μgmL in thepooled sera from diabetic patients and ± μgmLin pooled sera from nondiabetic individuals The diabetic serum induced a 3fold increase in PDA cell proliferation compared to the nondiabetic serum This effectwas greatly reduced by prior selective AGE removalfrom the diabetic serum AGE levels ± μgmLand almost completely reversed by combining AGE removal from serum and FL92616 treatment of PDAcells Fig DiscussionDespite the epidemiological evidence of increased PaCrisk in both type [] and [ ] diabetestheunderlying mechanisms still remains to be elucidatedHere we showed that STZinduced type diabeteswhich is characterized by marked hyperglycaemia andinsulin ia without weight gain [] significantlyaccelerated tumour progression in a mouse model ofKrasdriven PaC The absence of obesity and insulinresistance argues in favour of the hypothesis that thePaCpromoting effect of diabetes is directly related tothe adverse effects of hyperglycaemiaIn additionRCS trapping and AGE inhibition by FL92616 efficiently prevented the acceleration of PanIN progression to invasive PaC induced by diabetes Thedifference in the incidence of PaC between the twodiabetic groups ie untreated and treated with FL occurred despite similar increases of bloodglucose levels supporting the conceptthat glucosemetabolites but not glucose per se were responsiblefor PaC promotion STZtreated mice that failed todevelop or reversed hyperglycaemia showed the samePaC incidence as the Ctr group thus ruling out aneffect of STZ on invasive PaC development in DiabmiceOur finding of an association between AGE accumulation and YAP induction in PaC in | 2 |
neutrophils account for of circulating leukocytes and are the ï¬rst immune cells recruitedto an ammatory site they play an important role in the innate immune response topathogens as patients with neutropenia are highly susceptible to bacterial and fungal infections neutrophils perform numerous functions that target microbes including phagocytosis the releaseof antimicrobial peptidesproteases and netosis interestingly neutrophils have garneredconsiderable interest for their emerging and prominent roles in modulating cancer growth andmetastatic progression the roles played by neutrophils in the cancer setting are diverse andcomplex leading to the concept of neutrophil heterogeneityplasticity and the notion that distinctneutrophil subsets might existgranulopoiesisdiï¬erentiationand mobilization of maturefrom the bone marrow intocirculation this process begins with the commitment of granulocytemonocyte myeloidsegmented neutrophilsregulatedprocessthatinvolvestheisatightlyedited bybrahm segaluniversity at buffalo united statesreviewed byye liuniversity of texas md andersoncancer center united statesconnie jackamancurtin university australiacorrespondencepeter m siegelpetersiegelmcgillcaspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in immunologyreceived may accepted july published august citationhsu be shen y and siegel pm neutrophils orchestrators of themalignant phenotypefront immunol 103389ï¬mmu202001778frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypegmps which progressthrough a series ofprogenitorsneutrophil progenitors myeloblast promyelocyte myelocytemetamyelocyte band cell untilthey become a matureneutrophil in cancer dysregulated granulopoiesis hasled to theidentiï¬cation of diï¬erent neutrophil subsets that play a role intumor progression preneus comprise a neutrophil precursorpopulation that retain their proliferative capacity and expandin the bone marrow and spleen of tumor bearing mice preneus diï¬erentiate into immature and mature neutrophilswith the former found to accumulate in growing tumors anearly stage committed unipotent neutrophil precursor nep hasalso been identiï¬ed and their adoptive transfer into humanizedmice promoted solid tumor growth by inhibiting t cellactivation two neutrophil subsets highdensity neutrophilshdns and lowdensity neutrophils ldns were identiï¬ed invarious tumor models by diï¬erential density centrifugation hdns represent mature segmented neutrophils whereas ldnscomprise a heterogeneous mixture of mature and immatureneutrophils increasing mobilization of ldns into theperipheral blood was associated with enhanced tumor growthand metastasis functionsand antitumorigenicin addition to the identiï¬cation of distinct neutrophil subsetsneutrophils exhibit plasticity in response to tumorderivedfactors in a manner similar to macrophages neutrophils havebeen classiï¬ed into two categories n1 and n2 to describetheir prorespectively in vivo evidence has shown that tumorassociatedneutrophils tans can change their function from a protumor phenotype n2 to an antitumor n1 phenotypewith the addition of a tgf inhibitor arguing that tgfis an important factor driving the n2 phenotype incontrast signals associated with an antitumor n1 phenotypeinclude type iinterferons and those propagated by themet receptor however this categorization is likelyto represent an oversimpliï¬cation of neutrophil diversityneutrophil polarization similar to macrophages could alsorepresent a continuum of diï¬erent neutrophil phenotypespresent in the tumor microenvironment these advancesregarding the degree of neutrophil heterogeneityplasticityobserved in the cancer setting have sparked an intense andrenewed interestin this cell population while there areongoing discussions in the ï¬eld regarding the relationshipsubsets we directbetween pmnmdscs and neutrophilto excellentthe readerthatfully discussthese we will brieï¬y discuss antitumorrelationshipsneutrophilreview will primarilyroles of neutrophils and neutrophildiscussassociated functionsgrowth andmetastatic progressionfunctions howeverin promotingthe recentreviewstumorthisantitumor neutrophil functionscan participateantitumorneutrophilsmechanisms thattumor growth or eliminate cancercells a wellstudied neutrophilassociated function isin a variety oflimittheir ability to generate reactive oxygen species ros tolimit tumor progression upon tumor cell contact mousederived neutrophils can release hydrogen peroxide to eliminatemetastatic cancer cells in vitro subsequentlyit wasdemonstrated that expression of trpm2 transient receptorpotential cation channel subfamily m2 on tumor cells increasedtheirsensitivity to neutrophilmediated h2o2dependentcytotoxicity this occurred through a mechanism that involveda transient increase in ca2 mobilization within cancer cells trpm2 upregulation in tumor cells occurred followingan epithelialtomesenchymaltransition emt and cancercells that have undergone an emt were more susceptible toneutrophilmediated killing more recently an interactionbetween the receptor for advanced glycation end productsrage which is expressed on tumor cells and cathepsin gpresent on murine neutrophils was shown to mediate in vitrotumor cell cytotoxicity in a h2o2dependent manner the release of neutrophil ros is also dependent on the tumormicroenvironment in hypoxic tumor microenvironments theability of murine neutrophils to kill tumor cells in vivo throughthe release of ros is greatly diminished thus neutrophilshave the capacity to mediate rosdependent direct tumorcell killingcausingthe interplay of neutrophils with otherimmune celltypes can also indirectly limittumor progression tumorassociated neutrophils suppress the protumorigenic role ofil17 secreting Îδ t cells by inhibiting their proliferationlow glutathione levels in Îδ17 t cells rendered them sensitiveto neutrophilderived rosenhanced oxidativestress and reduced proliferation in earlystage humanlung cancer a subset ofimmature neutrophils have beenidentiï¬ed as having antigenpresenting functions and act topromote antitumor immunity by stimulating the secretionofinaddition to neutrophilt cellinteractions communicationbetween neutrophils and monocytes can also elicit antitumoreï¬ects nonmetastaticifnÎproducing monocytes to the lungs ifnÎ release activatestmem173sting within neutrophils whichstimulatesneutrophilmediated killing of disseminated cancer cells in thelungs ammatory cytokinesfrom t lymphocytescan mobilizecancercellsneutrophils have been shown to ltrate deposits of prostatecancer cells within bone metastases importantly neutrophilsimpaired bone metastasis progression by inhibiting stat5signal transducer and activator of transcription functionwithin prostate cancer cells resulting in their apoptotic cell death recently neutrophils have been reported to be involvedin antibodymediated trogocytosis a process that mechanicallydisrupts the plasma membrane of antibodyopsinized cancercells leading to a lyticnecrotictype cell death iga antibodiesagainst receptors expressed by cancer cells her2 egfr couldenhance neutrophilmediated trogocytosis of cancer cells if thecd47sirpα innate immune cell checkpoint was simultaneouslyblocked taken together these results demonstrate thatneutrophils can impair tumor growth and metastasis using acombination of direct and indirect cancer cell killing mechanismssupplementary table frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure neutrophil functions that promote tumor growth and metastasis to support primary tumor growth neutrophils can mediate t cell suppression and altermacrophage differentiation neutrophil release of timp1 enhances tumor cell invasion by inducing epithelialtomesenchymal transition once in circulation circulatingtumor cells interact with neutrophils which enables tumor cell proliferation secretion of various proammatory markers such as il8 il1 or mmps can mediateincreased tumor cell extravasation in addition neutrophils can inhibit intraluminal nkmediated killing of circulating cancer cells leading to increased extravasation atthe metastatic site various systemic and microenvironmental factors can promote neutrophil ltration neutrophils can awaken dormant cancer cells by promotingecm remodeling and angiogenesis lastly continued growth of the metastatic lesion is facilitated by key neutrophildependent mechanisms which includeangiogenesis proliferation immune suppression and immune exclusion csf1 colony stimulating factor timp1 tissue inhibitor of matrix metalloprotease pdl1programmed death ligand tgf transforming growth factor ros reactive oxygen species mmp matrix metalloproteinases gmcsf granulocyte macrophagecolony stimulating factor angptl2 angiopoetin like2 fgf2 ï¬broblast growth factor ltb4 leukotriene b4 inos inducible nitric oxide synthase net neutrophilextracellular trap caf cancerassociated ï¬broblastneutrophil functions thatpromote primary tumor growthneutrophils promote primary tumor growth by variousmechanisms figure netosis is a process that involvesthe extrusion of neutrophilderived chromatin structures thatare decorated with neutrophil granule constituents whichform extracellular structures called neutrophil extracellulartraps nets normally netosis and net productionhave been described in the context of a neutrophils ability tocapture and kill bacteria extracellularly however netshave been shown to play an important role in the growth of aprimary tumor tumor microenvironmental changes includingtumorassociated coagulation and enhanced thrombosishave been linked to enhanced tumor growth several recentstudies suggest that netosis may play an important role inthese processes lps stimulation was shown to increase c3arexpression within neutrophils enhance netosis and increasecoagulation these events were correlated with n2 neutrophilpolarization and increased tumor growth interestingly ithas recently been shown that immature neutrophils preferentiallyrespond to cancer cell derived c3a to promote their migration subsequently it was shown that breast cancer cells thatexpressed high levels of gcsf and il1 exhibited highneutrophil counts and tumorassociated thrombosis which wasdependent on net formation pharmacological blockadefrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeof il1 receptor signaling reduced net formation attenuatedtumorassociated thrombosis and impaired tumor growth nets can also directly uence cancer cell proliferationneutrophil elastase ne present within nets activates tumorcells to increase mitochondria biogenesis and atp productionthereby further enhancing the growth of cancer cells in addition to the impact of nets neutrophils canalso interact with other immune cells through additionalmechanisms to promote tumor growth neutrophilderived roscan inhibit t cell proliferation creating an immunosuppressiveenvironment that is supportive of tumor growth phenotypiccharacterization and singlecell rna sequencing identiï¬ed aneutrophil subset that is cd84hi which exhibited potent t cellsuppressive activity and increased ros production in amodel of gastric cancer neutrophils were activated by tumorderived gmcsf that resulted in elevated programmed deathligand pdl1 expression these pdl1 neutrophils wereable to suppress t cell function and promote tumor growth secretion of mmp9 matrix metalloproteinase fromltrating neutrophils activates latent tgf and induces tcell suppression and tumor growth in a colorectal cancer model siglecfhigh neutrophils in lung adenocarcinoma createdan immunosuppressive environment by promoting macrophagediï¬erentiation causing the release of high levels of rosand enabling tumor progression together these ï¬ndingsindicate that neutrophils that ltrate diverse primary tumorscan modify the local environment in diï¬erent ways to favortumor growthneutrophil functions thatpromote metastasisthe ability of cancer cells to leave the primary tumor anddisseminate to distant ans represents the deadliest aspectof cancer progression indeed the emergence of metastaticcancer accounts for ¼ of cancer related deaths themetastatic cascade represents a series of barriers to cancercells and neutrophils have been found to assist cancer cells insuccessfully navigating several of these distinct steps figure supplementary table local invasionintravasationltrating neutrophils within primary tumors are associatedwith an increase in emt enhanced metastasis and pooroutcomes mechanisticallyof matrixmetalloprotease timp1 secreted by neutrophils induced anemt and consequently increased the migration and invasion oftumor cells cancer cells that had undergone an emt expressedcd90 which enhanced timp1 secretion by neutrophils in acontactdependent manner inhibitortissuesurvival in circulationextravasationthe ability of circulating tumor cells ctcsto surviveis criticalfor metastasis formation the formation ofheterotypic cancer cellneutrophil clusters was found to greatlyincrease metastatic ï¬tness using a 4t1 breast cancer modelit was demonstrated that ctcneutrophil interactions reliedon vcam1 dependent adhesion which enhanced cancercell proliferation and increased metastasis indirectlyneutrophils can also inhibit nk cellmediated tumor clearancein circulation thereby increasing the intraluminal survival ofdisseminated tumor cells in this study 4t1 breast cancer cellswere injected subcutaneously to mobilize murine neutrophilsly6gfollowing which d2a1 breast cancer cells wereinjected intravenously mice bearing 4t1 cells exhibited reducedclearance of d2a1 cells from the lungs when compared to micethat were not injected with 4t1 cells depletion of nk cellsresulted in enhanced d2a1 cancer cell accumulation in the lungswhile neutrophil depletion had the opposite eï¬ect cancer cells that have survived in circulation must exitthe bloodstream and extravasate into tissue parenchyma neutrophils have been shown to regulate the extravasationprocessthrough several mechanisms neutrophilderivedfactors can diminish the integrity of the endothelial barrierpermitting cancer cellsil8il1 and matrix metalloproteasesmmp8 and mmp9released from neutrophils activated endothelial cells reducedendothelialtransendothelialmigration and accelerated the rate of cancer cell extravasation to extravasate more easilyincreasedfunctionbarriersitesnetosis and net constituents can support cancer cellextravasation through enhanced trapping of ctcs withinmetastaticimportantly blocking netosisdecreases cancer cell adhesion and inhibits metastatic spread tothe lung and liver furthermore changes within speciï¬cmetastatic microenvironments such as exposure to ozoneor redox imbalance triggered netosis and led to increasedentrapment of cancer cells in the lung and enhanced metastasis collectively these studies show that neutrophils play animportant role in enhancing tumor cell survival and increasedextravasation which promote cancer metastasisrecruitmentearly seedingsurvivalsystemic and tumorderived factors have been implicatedin neutrophilin the premetastatic nichetumorderived il1 induces Îδ t cells to produce il17aand granulocytecolony stimulating factor gcsf whichresults in the recruitment of immunosuppressive neutrophilsto the lung gmcsf and il5 have been shown topromote the expansion and recruitment of prometastaticneutrophils in the lungs of obese mice which promotes lungmetastasis angiopoetinlike2 angptl2 secreted byosteosarcoma cells implanted in the tibia stimulates lungepithelial cells which led to the accumulation of neutrophilsin the lung and enhanced lung metastatic burden in the lung neutrophils secrete ltb4increases theinitiating cellsproliferation of ltb4rpositive metastasis activation of notch1 in colorectalcellsdrives tgf2dependent recruitment of immunosuppressiveneutrophils within the liver which enabled the formation of livermetastases cancerthatnets also support early cancer cell seeding and colonizationof metastases induction of nets by ovarian tumorderivedfactors has been shown to be important in promoting metastasisfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeto the omentum in the liver nets have also beenshown to promote metastasis by activating cancerassociatedï¬broblasts growth in the metastatic siteneutrophils have been shown to promote the growth ofmetastases after seeding minor subclones of breast cancer cellsthat secrete il11 and figf cfosinduced growth factor cansupport the formation of polyclonal metastases composed ofdriver and passenger subpopulations these il11 producingsubclones activated il11 responsive mesenchymal stromal cellswhich induced chemokine secretion and subsequent recruitmentof prometastatic neutrophils tumor cellderived gmcsfwas shown to stimulate neutrophils to synthesize and secretetransferrin an iron transport protein which has mitogenicactivity that promotes lung metastatic growth when taken up bycancer cells a recurring function of prometastatic neutrophils is theirability to create an immunosuppressive microenvironmentthat support metastasis within lung metastasesinduciblenitric oxide synthase inos producing neutrophils havebeen shown to limit cd8 t cell dependent antitumorresponses by promoting immune suppression recently p53deï¬cient cancer cells were found to increase the expressionof wnt ligands which in turn upregulated il1 productionfrom tumorassociated macrophages high il1 levelsengaged Îδ17 t cells which subsequently enhanced neutrophilrecruitment that promoted the formation of lung metastases furthermore loss of elf5 e74like transcription factorexpression in triplenegative breast cancer led to increased ifnÎ signaling resulting in the expansion of immunosuppressiveneutrophils in addition to tumorderived factors a lackof systemic testosterone levels can lead to an impairmentof antitumor neutrophil functions a shift toward immatureneutrophils was observed in castrated male mice leading toincreased neutrophilderived ros and suppression of nk cellactivation that promoted increased lung metastatic burden intwo melanoma models recently a role for net formationhas been described for the continued growth of establishedmetastases nets released during cancer progressionwas shown to limitthe ability of nk and cytotoxic tcells to eliminate cancer cells speciï¬cally net formationimpaired direct contact between the cancer cells and cytotoxicimmune cells nk and t cells inhibition of netosis with aprotein arginine deiminase pad4 inhibitor synergized withimmune checkpoint inhibitors to control tumor growth andmetastasis proangiogenic functions have long been ascribed forneutrophils which revealed that neutrophilderived proteasessuch as mmp9 could release stored angiogenic factors vegffgfs that were stored in the local environment to enable bloodvessel formation recently a diï¬erent mechanism bywhich neutrophils enhance angiogenesis has been describedthe synthesis and secretion of ï¬broblast growth factor fgf2 by neutrophils in the liver microenvironment drivesangiogenesis and growth of nascent colorectal cancerderivedhepatic metastases dormantresidual disease andtherapy resistanceneutrophils have also been implicated in awakening dormantcancer cells lpsinduced tissue ammation led to metastaticoutgrowth of dormant tumor cells in a neutrophildependentmanner mmp9 produced by neutrophils can trigger thegrowth of dormant cancer cells by remodeling extracellularmatrix and releasing potent angiogenic factors ne andmmp9 which are enzymes associated with nets can cleavethe extracellular matrix ecm leading to integrinmediatedsignaling which awakens dormant cancer cells and promotescancer cell growth severalstudies have shown that neutrophils promoteresistance to therapy doxorubicin and paclitaxel resistant breastcancer cells express more il17 and cxcr2 ligands whichincreases neutrophil recruitment a neutrophilenrichedsubtype characterized in triple negative breast cancer tnbcdetermined that neutrophils were largely immunosuppressiverendering these tumors resistant to immune checkpoint blockadetherapy in a genetically engineered mouse model ofsarcoma neutrophils promote resistance to radiation therapyby activating mitogenactivated protein kinase mapk pathway in addition cd177 neutrophil ltrates in colorectalcancer patients are associated with adverse outcome in patientsreceiving bevacizumab [antivascular endothelial growth factora vegf a] furthermore lysyl oxidaselike loxl4expressing neutrophils that ltrated colorectal cancer livermetastases were found to identify patients that were resistant toantiangiogenic therapy metabolic programming inneutrophilsrecentinteresthasbeenconceptin thethereofimmunometabolism and the realization that altered cellularmetabolism in ltrating immune cells can have a signiï¬cantimpact on tumor growth and metastasis neutrophilsare typically viewed as a cell type that is heavily reliant onglycolysis to perform their eï¬ector functions consistentwith this notion neutrophils have very few mitochondria andinhibitors of oxidative phosphorylation oxphos do notalter their rates of oxygen consumption howeverduring tumor progression neutrophils have been shown toundergo a metabolic switch which involves the upregulationof genes associated with oxphos fatty acid metabolism andglycolysis figure neutrophils isolated from lewis lungcarcinoma exhibit increased ï¬ux through oxphos glycolysisand increased atp production compared to naïve neutrophilssuggesting that multiple metabolic strategies are engaged intumor ltrating neutrophils recently upregulation offatp2 fatty acid transport protein in neutrophils was shownto increase lipid accumulation in these cells fatp2 regulated theuptake of arachidonic acid which was subsequently convertedto prostaglandin e2 neutrophilderived prostaglandin e2 wasfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure metabolic changes in cancerassociated neutrophils neutrophils which possess few mitochondria are reliant on glycolysis to generate atp to fueleffector functions such as phagocytosis generation of reactive oxygen species and netosis in cancer neutrophils upregulate oxidative phosphorylation oxphosand fatty acid transporters to mediate many neutrophil functions including migration and t cell suppression under nutrient limiting conditions such as low glucoseneutrophils can reprogram their metabolism to break down fatty acids or utilize certain amino acids glutamate proline to fuel protumorigenicprometastaticfunctions ppp pentose phosphate pathway glut glucose transporter mct monocarboxylate transporter tca tricarboxylic acid cycle fatp2 fatty acidtransport protein aa arachidonic acid pge2 prostaglandin e2found to be important or neutrophilmediated cd8 t cellsuppression and tumor growth metabolic ï¬exibility refers to the ability of a cell to shiftbetween one metabolic program to another in response tochanging metabolic demands or nutrient supply high metabolicï¬exibility increases the cells ability to survive various andeverchanging metabolic microenvironments neutrophilsubpopulations can also exhibit metabolic ï¬exibility figure in breast cancer splenic neutrophils can engage mitochondrialdependent fatty acid oxidation as a predominate fuel sourceto support ros production and maintain t cell suppression under glucoselimiting conditions similar to certain tumormicroenvironments immature ldns have been shown to utilizeoxphos to generate atp that is required to support theirprotumorigenic functions indeed immature ldns can supportnetosis under nutrient limiting conditions via mitochondrialdependent amino acid catabolism which is importantforeï¬cient breast cancer liver metastasis in addition thelongevity of neutrophils could also be altered due to the enhancedmetabolic ï¬exibility the ex vivo halflife of mouse circulatinghdns and ldns was and h respectively suchobservations raise the intriguing possibility that under certainconditions distinct neutrophil subsets may not be as shortlived as previously thought these studies argue that increasedmetabolic ï¬exibility in distinct neutrophil populations may beimportant for cellular functions that can uence tumor growthand metastatic progressionclinical importance futureperspectives on treatmentin keeping with their protumorigenicmetastatic functions thepresence of neutrophils across diï¬erent cancers was shownto be strongly associated with adverse patient outcomes frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeamong certain subtypes of breast cancer er the presence ofa neutrophil ltrate in the primary tumor is also indicativeof worse patient outcomes furthermore in patients withadvanced cancers serum il8 levels and neutrophil ltrationare associated with worse overall survival and diminishedresponse to immune checkpoint inhibitors the mobilization of neutrophils into circulation also hasprognostic signiï¬cance the neutrophiltolymphocyte rationlr is an important risk stratiï¬cation and treatment selectiondiagnostic tool for cancer patients a high nlr is associated withpoor prognosis in many solid human cancers a highnlr is also associated with decreased overall survival in patientswith tnbc or metastatic breast cancer an important and unanswered question with respect to thenlr is the type of neutrophil that is being detected in thesepatients are they high or lowdensity neutrophils interestinglyldns have been identiï¬ed in patients with breast cancer lungcancer head and neck cancers urologic cancers and lymphoma in patients with advanced lung cancerit wasreported that higher proportion of ldns predictedpoorer survival these observations are in keeping withthe protumorigenic and prometastatic functions associatedwith ldnn2 neutrophils while most studies reveal a negativeprognostic impact of neutrophils in cancer there was one studythat associated the presence of a cd16high cd62dim neutrophilsubset with increased survival of head and neck squamous cellcarcinoma patients these observations highlight the needfor better markers that are capable of discriminating betweenneutrophils that exert antitumor vs those that mediate protumormetastatic eï¬ectsmechanistic insights have greatly advanced our knowledgeoftumorderived factorstumor growth andmetastasis in a neutrophildependent manner additional studiesimpactthatreferences sipsas nv bodey gp kontoyiannis dp perspectives for the management offebrile neutropenic patients with cancer in the 21st century cancer 101002cncr20890 kolaczkowska e kubes p neutrophil recruitment and function in healthand ammation nat rev immunol 101038nri3399inde visser ke neutrophilssb wellenstein md coï¬eltcancer neutral no more nat rev cancer 101038nrc201652 cowland jb borregaard n granulopoiesis and granules of humanneutrophils immunol rev 101111imr12440 evrard m kwok iwh chong sz teng kww becht e chenbone marrow neutrophilstraï¬ckinganalysisspecializedpopulationsexpansioninofal developmentaljetrevealsand 101016jimmuni201802002functionseï¬ectorimmunity79e8 zhu yp padgett l dinh hq marcovecchio p blatchley a wu r identiï¬cation of an early unipotent neutrophil progenitor with pro tumoralactivity in mouse and human bone marrow cell rep 41e8 101016jcelrep201807097 sagiv jy michaeli j assi s mishalian i kisos h levy l phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancercell rep 101016jcelrep201412039focused on characterizing the phenotypic and functional role ofneutrophils in cancer it may be possible to develop strategies thatspeciï¬cally target those neutrophil subsets that actively promotetumor growth and metastasis while sparing those neutrophilsthat possess antitumor and antimicrobial functions finallythe emerging concept of metabolic ï¬exibility that is exhibited bycertain neutrophil subsets may aï¬ord new ways of targeting theseprotumorigenicmetastatic neutrophilsauthor contributionsbh ys and ps wrote the review and prepared the ï¬guresall authors contributed to the and approved thesubmitted versionfundingwork from the authors laboratory cited in this review wassupported by an operating grant to ps from the cancer researchsociety and the terry fox research institute and québecbreast cancer foundation grant bh acknowledgessupport from the charlotte and leo karassik foundation phdfellowship and the rolande and marcel gosselin graduatestudentship ys holds an entrance studentship from thegoodman cancer research centre ps is a mcgill universitywilliam dawson scholarsupplementary materialthe supplementary materialfor this can be foundonline at httpswwwfrontiersins103389ï¬mmu202001778fullsupplementarymaterial coï¬elt sb kersten k doornebal cw weiden j vrijland k hau cs il17producing gammadelta t cells and neutrophils conspireto promote breast 101038nature14282cancer metastasis nature hsu be tabaries s johnson rm andrzejewski s senecal j lehuede c immature lowdensity neutrophils exhibit metabolic ï¬exibility thatfacilitates breast cancer liver metastasis cell rep 15e6 101016jcelrep201905091 fridlender zg sun j kim s kapoor v cheng g ling l polarizationof tumorassociated neutrophil phenotype by tgfbeta n1 versus n2tan cancer cell 101016jccr200906017 ohms m möller s laskay t an attempt to polarize human neutrophilstoward n1 and n2 phenotypes in vitro front immunol 103389ï¬mmu202000532jablonska j leschner s westphal k lienenklaus s weiss s neutrophilsresponsive to endogenous ifnbeta regulate tumor angiogenesis andgrowth in a mouse tumor model j clin invest 101172jci37223 finisguerra v di conza g di matteo m serneels j costa s thompsonaa met is required for the recruitment of antitumoural neutrophilsnature 101038nature14407 ostuni r kratochvill f murray pj natoli g macrophages and cancer frommechanisms to therapeutic implications trends immunol 101016jit201502004frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotype brandau s moses k lang s the kinship of neutrophils and granulocyticmyeloidderived suppressor cells in cancer cousins siblings or twins semincancer biol 101016jsemcancer201302007 vols s sionov rv granot z always look on the bright side antitumor functions of neutrophils curr pharmac design granot z henke e comen ea king ta norton l benezra r tumorentrained neutrophils inhibit seeding in the premetastatic lung cancer cell 101016jccr201108012 gershkovitz m caspi y fainsodlevi t katz b michaeli j khawaled s trpm2 mediates neutrophil killing of disseminated tumor cells cancer res 10115800085472can173614 gershkovitz m fainsodlevi t khawaled s shaul me sionov rvcohendaniel l microenvironmental cues determine tumor cellsusceptibility to neutrophil cytotoxicity cancer res 10115800085472can180540 sionov rv fainsodlevi t zelter t polyansky l pham ct granotz neutrophil cathepsin g and tumor cell rage facilitate neutrophilanti8e1624129 1010802162402x20191624129cytotoxicity oncoimmunologytumor mahiddine k blaisdell a ma s crequergrandhomme a lowell caerlebacher a relief of tumor hypoxia unleashes the tumoricidal potentialof neutrophils j clin invest 101172jci130952 mensurado s rei m lanca t ioannou m goncalvessousa n kubo h etal tumorassociated neutrophils suppress protumoral il17 gammadeltat cells through induction of oxidative stress plos biol 16e2004990 101371 pbio2004990 singhal s bhojnagarwala ps obrien s moon ek garfall al rao as etal origin and role of a subset of tumorassociated neutrophils with antigenpresenting cell features in earlystage human lung cancer cancer cell 101016jccell201606001et hagerling c gonzalez h salari k wang cy lin c roblescooperationtumor prevents metastatic progressionaliinduced byof breast cancer proc natl acad sci usa 101073pnas1907660116eï¬ector monocyteneutrophilimmunethe primary costanzogarvey dl keeley t case aj watson gf alsamraae myu y neutrophils are mediators of metastatic p | 0 |
"ammatory bowel disease ibd is a long life disease with remission and relapse periods ibd arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers in addition ibd has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] ulcerative colitisuc and crohn's diseases cd are known as two main forms of ibdaccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized in this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way for example 5asa which is acommon drug in the treatment of ibd is less eï¬ective on maintainingremission in cd patients on the other hand antibiotic therapy is notrecommended for the treatment uc but it can be eï¬ective on cd patients diï¬erential diagnosis is a serious challenge because cdand uc have significant similarities in terms of their clinical endoscopic and histological features however there are some diï¬erencesbetween uc and cd which are summarized in table1 in addition tointestinal complications uc and cd also have significant extraintestinal manifestations for example it was shown that uc is significantly associated with primary sclerosing cholangitis and cd is alsoassociated with cholelithiasis especially in cases that the ileum is involved furthermore cd can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections both cd and uc can cause several disorderssuch as arthritis erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of ibd the latest statistics showed that the global corresponding author at department of clinical biochemistry and laboratory medicine faculty of medicine tabriz university of medical sciences daneshgahstreet po box tabriz iranemail address vagharimtbzmedacir m vagharitabari101016jcca202008025received july received in revised form august accepted august available online august elsevier bv all rights reserved 0cf khakikhatibi table1clinical endoscopic and histological features of cd and ucclinical featuresfeaturesrectal bleedingabdominal painfevermucus defectionintestinal obstructionperineal diseasepostoperative recurrenceasca positiveanca positiveendoscopic featurescdoccasionallyfrequentlyfrequentlyoccasionallyyesyesyesfrequentlynot commonucfrequentlyoccasionallynot commonfrequentlynonononot commonfrequentlyfeaturescduclocationmucosal involvementdepth of ulcerationï¬stulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperï¬cialnonooccasionallyfrequentlyucrarefrequentlynot commonprevalence of ibd currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem according to areport published in ibd has the highest prevalence rate ineurope and its prevalence in the newly industrialized countries of asiaafrica and south america also appears to be increased over the pastthree decades unfortunately the peak of the disease is at the young age of years old therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societymoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease the invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage ibd which are unpleasant for patients as well as having thehigh associated costs now the gold standard method for diagnosingibd and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring ibd patients so in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of ibd the role of fecal calprotectin in diagnosis and management ofibdthe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of ibd diagnosis and management have been studied including ibd diï¬erentiation from irritable bowel syndrome ibs evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andclinica chimica acta response to treatment in following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas calprotectin a clinically valuable proteincalprotectin is an antimicrobial protein mainly secreted by neutrophils this protein competes with bacteria over zinc thus kills thebacteria however this is not the only contribution that it has to antimicrobial activity moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsserum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection at theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints in addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker in neonatal sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of neonatal sepsis it has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low crp levels so they appear to be more eï¬cient at reï¬ecting disease activity some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof ibd impressive results have been reported a study onpatients with ibd both uc and cd have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in ibd diagnosis compared to crp and albumin this studyalso indicated that the combination of serum calprotectin with crp oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with cd however no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with cd and uc as well as a slight correlation betweenserum calprotectin level and crp that was observed only in patientswith uc another study showed that the serum level of calprotectin was significantly higher in patients with cd compared to healthyindividuals in addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease the eï¬cacy of salivary calprotectin in the diagnosisof ibd has also been studied which showed that salivary calprotectinsignificantly increased in patients with ibd compared to healthy individuals in this study auc values for unstimulated saliva and stimulated saliva to distinguish ibd patients from healthy individualswere reported to be and respectively however thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of ibd that is discussed in the following 0cf khakikhatibi clinica chimica acta laboratory measurement and reference intervalfecal calprotectin is a stable protein that remains stable for daysat room temperature this property is an excellent advantage for alaboratory marker also it seems that keeping the specimen at refrigerated temperature °c can increase the stability of fecal calprotectin however evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature on the other hand it is not also recommended tokeep samples in the refrigerator for more than days it seemsthat fecal calprotectin remains stable up to one year at °c measurement of fecal calprotectin can be done both qualitatively andquantitatively accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette however in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only it seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom ibd patients rapidly however some studies have shown that it isnot accurate enough in this case as well nevertheless asignificant concordance has been reported between home test resultsibdoc and fecal calprotectin laboratory measurement results whenquantum blue calprotectin elisa kit was used notably the agreements between results were and depending on the selectedcutoï¬s several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin these tests reportpositive results ranged from to µgg there are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin these kits are usually designed in terms of the elisamethod and some have a measurement range between and µgg moreover the chemiluminescence immunoassays cliamethod can also detect values between and µgg fluoro enzyme immunoassays feia and particle enhanced turbidimetric immunoassays petia can also be used for the measurement of fecalcalprotectin in this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals among healthy adults there is asignificant agreement on µgg as an upper limit one study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric ibd table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies according to these reports age can aï¬ect fecal calprotectinlevels fecal calprotectin and ibd diagnosisonly a small percentage of patients complaining of abdominal painand diarrhea have ibd in many cases ibs as a functional gastrointestinal disorder is known as the cause of such clinical symptomspatients with ibs have normal colonoscopy results while ibd patientsindicate abnormal colonoscopy results and have intestinal ulcersunfortunately the significant prevalence of ibs and the overlap between clinical symptoms and ibd can increase the colonoscopy ratetherefore a noninvasive diagnostic marker can be very helpful in thisregard notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of ibd was obtained in the 1990s røseth in proposed a method for measuring calprotectin in stool specimens one of the ï¬rst and most interesting studies regarding fecal calprotectinutility in ibd diagnosis was the study by røseth published in in this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls this study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study in another study published in auc values of ci were reported for fecal calprotectin in thediagnosis of colorectal ammation moreover in a study onchildren with ibd it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with esr levels in astudy published in kolho reported auc values of ci for fecal calprotectin in the diagnosis of pediatric ibd in a study on patients with crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease the results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as crp andesr in the diagnosis of ibd diamanti reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of ibd however oursample size was and the majority of patients were in the active phaseof the disease in another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard in one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withibd from patients without ibd patients with diseases other than ibdpatients with ibs and healthy persons with sensitivity and speciï¬city caviglia in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between ibs and ibd howeversome studies have reported significantly lower values accordingly in astudy on patients with ulcerative colitis kalantari reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with crohn's disease asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce ï¬ndings anddiagnosis of crohn's disease in another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city oftable reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesagesmedian levels of fecal calprotectin range µggnumber of subjectsused kitup to monthchildren yearschildren yearsadultsover years bühlmann elisabühlmann elisacalpro® calprotectin elisa test alpphicalphicalreference 0cf khakikhatibi were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in crohn's disease given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of ibd where the small intestine is involvedbesides there are some preanalytical limitations which are explainedin the next sections therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenibd and ibs the probability of ibd was less than at fecal calprotectin values lower than µgg or crp values lower than mgdl therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of ibd in patients with ibslike symptoms aswell as reducing the rate of colonoscopy moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between ibdand ibs it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points hence performing extensive studies indiï¬erent countries on the healthy population and the ibd patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultstable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of ibdfrom ibs and table4 summarizes some metaanalysis results in thisregard as shown in table the most important limitation of the majority of clinical studies conducted to date is the small sample size alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between ibdand nonibd diseases fecal calprotectin and endoscopic and histologic activity evaluationundoubtedly one of the most serious challenges in the managementof ibd is evaluating the endoscopic and histologic activities of thedisease nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withibd as noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations in recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels in addition many studies have been performed inthe last decade all of which cannot be reviewed in this article the ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the ï¬rst studiesroseth found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis furthermore in another study they observed that ibdpatients who were in remission clinically and had normal fecal calprotectin levels less than mgl had normal colonoscopy results these interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andclinica chimica acta table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdsample sizepooled sensitivitypooled speciï¬cityreferences mucosal healing in ibd patients also these studies were the startingpoint of extensive studies that have been conducted up to now in astudy conducted on patients with crohn's disease sipponen investigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of crohn's disease correspondinglythe researchers used the crohn's disease endoscopic index of severitycdeis scoring system in their study to evaluate the endoscopic activity of crohn's disease as a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and speciï¬city in another study cdeis and mayo disease activity indexmdai were used to evaluate the endoscopic activity of crohn's diseaseand ulcerative colitis respectively according to the results of thatstudy on ibd patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to therachmilewitz clinical activity index in addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like crp besides one studyfound that fecal calprotectin was more strongly correlated with thesimple endoscopic score for crohn's disease sescd compared to thecrp and even crohn's disease activity index cdai the modiï¬edbaron index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis as a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to crp and clinical activity of the disease in thisregard similar results were also observed in our recent study in whichthe ulcerative colitis endoscopic index of severity uceis and sescdwere used therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of ibd endoscopicactivity the high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring however severalrecent studies have reported some significantly lower values accordingly in a recent study in which mayo endoscopic score [mes] wasused to evaluate the endoscopic activity of ulcerative colitis atable summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with ibd and without ibdnumber of ibd patientsage grouplocationcut oï¬sensitivityspeciï¬city cd and uc cd and uc cd and uc and unclassiï¬ed68cd and uc cd and uc and unclassiï¬ed cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsreï¬dbib60 0cf khakikhatibi clinica chimica acta table summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in ibd patientsage groupstudylocationusedendoscopicactivity indexcorrelationcoeï¬cientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modiï¬edcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating mes ¤ in patients with ulcerative colitis were and respectively overall as presented in table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and ibd endoscopic activity although some of these studies reported a strong correlation some others reported a relativelyweak correlation as noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of ibd undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences however fecal calprotectin does not appear to be a very reliable marker for the predictionof ibd endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy inthis regard further studies are still needed however under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate ibd endoscopic activity can be helpfulpregnant patients with ibd have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy in one study physicianglobal assessment [pga] which is a clinical symptombased criterionwas used to evaluate ibd activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with ibd the results of this study showed a significantcorrelation between fecal calprotectin and pga levels at prepregnancyduring pregnancy and postpartum stages in another study asignificant association was reported between fecal calprotectin levelsand clinical activity of ibd in pregnant women moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also conï¬rmed the conclusions obtained from these studies according tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith ibd clinical activity during pregnancy therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of ibd fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of ibd endoscopic activity inpregnant women in addition under pandemic conditions fecal calprotectin can be very helpful following the covid19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy therefore noninvasive ibd management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before the combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringcovid19 pandemic therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for ibd endoscopic activity evaluation during pandemic fecal calprotectin appears to be associated with ibd histologic activity as well given thediï¬culty in the evaluation of the histologic activity of crohn's disease some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far correspondingly thesesystems score the disease's histologic activity based on histologic observationstherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory in this regard one of these histologic scoring systems isroberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the roberts scoring system theede also used themodiï¬ed harpaz index and performed some interesting studies in thisregard in one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingauc ci95 sensitivity speciï¬city andcutoï¬ mgkg in another study on patients with endoscopically inactive ulcerative colitis mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg p also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg in a recent study the geboes index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand speciï¬city for fecal calprotectin in the prediction of geboesscore sensitivity speciï¬city and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low speciï¬city were ï¬nally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand speciï¬city for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a speciï¬city of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconï¬icts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta challenges in ibd management the ï¬rst evidence of the eï¬cacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the ï¬rststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a speciï¬city of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and speciï¬city of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a speciï¬city of versus a sensitivity of and a speciï¬city at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and speciï¬city rates for fecal calprotectin in the prediction of relapse a sensitivity of and a speciï¬city at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and ï¬nally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas | 0 |
the hypoxic tumour is a chaotic landscape of struggle and adaption against the adversity of oxygen starvationhypoxic cancer cells initiate a reprogramming of transcriptional activities allowing for survival metastasis andtreatment failure this makes hypoxia a crucial feature of aggressive tumours its importance to cancer and otherdiseases was recognised by the award of the nobel prize in physiology or medicine for research contributing toour understanding of the cellular response to oxygen deprivation for cancers with limited treatment options forexample those that rely heavily on radiotherapy the results of hypoxic adaption are particularly restrictive to treatmentsuccess a fundamental aspect of this hypoxic reprogramming with direct relevance to radioresistance is the alterationto the dna damage response a complex set of intermingling processes that guide the cell for good or for badtowards dna repair or cell death these alterations compounded by the fact that oxygen is required to inducedamage to dna during radiotherapy means that hypoxia represents a persistent obstacle in the treatment of manysolid tumours considerable research has been done to reverse correct or diminish hypoxias power over successfultreatment though many clinical trials have been performed or are ongoing particularly in the context of imagingstudies and biomarker discovery this research has yet to inform clinical practice indeed the only hypoxia interventionincorporated into standard of care is the use of the hypoxiaactivated prodrug nimorazole for head and neck cancerpatients in denmark decades of research have allowed us to build a picture of the shift in the dna repair capabilitiesof hypoxic cancer cells a literature consensus tells us that key signal transducers of this response are upregulatedwhere repair proteins are downregulated however a complete understanding of how these alterations lead toradioresistance is yet to comefacts hypoxia is present in almost every solid tumour hypoxia is a major barrier to effective radiotherapyand is associated with radioresistance the hypoxic tumour is highly heterogenous withregions of chronic and acute hypoxia altered ph andimmune ltration differences in gene expression and protein functioncan occur between acute or chronic and mild orsevere hypoxiacorrespondence mahvash tavassoli mahvashtavassolikclacuk1head and neck oncology group centre for host microbiome interactionkings college london hodgkin building london se1 1ul ukedited by i amelio all dna damageresponsehomologousddr pathwaysincludingnonhomologous end joining missmatch repair andthe fanconi anaemia pathways have been shown tosuffer alterations in hypoxiarecombination activation of ddr transducer protein atm is seenin severe hypoxia in the absence of classical atmactivatingstranddna breaksfeaturesdoublesuchas atr is also activated most likely in response tohypoxiainduced replication stress however downregulation of dna repair effectorproteins such as rad51 and brca12 is seen results of ddr reprogramming include geneticinstability aberrant cell cycle and apoptotic control the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40ofï¬cial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of open questions precisely how do alterations to the ddr in hypoxialead to radioresistance for example when genomicinstability and generation of radioresistant clonestakes several cell divisions to set in how does adecrease in dna repair ability lead to increasedradioresistance what aspects of the hypoxic response could betargeted to radiosensitise or more effectively treattumours particularly in the context of ddr forexampleupregulated ddrtransducers such as atm atr and dnapkcs orinduce synthetic lethality following downregulationof dna repair effectorscan wetarget do different types of cancers have different patternsof ddr alteration within hypoxic tumours thisparticularly needs further research as tissues havebeen shown to have different oxygen pressureslevels of hypoxia and hypoxic heterogeneity can we use the data on this subject to develop abiomarkerhypoxiainducedradioresistance as we have done using hypoxia as asingle parametersignatureof how can we monitor hypoxic tumours during thecourse of a patients disease to help guide treatmentand how can wereportingreliableensureinterpretation of in vitro and in vivo dataintroductionofprogramstranscriptionalhypoxia is present in almost every solid tumour aninevitability of cancers characteristic disanised andfunctionally inefï¬cient vasculature rapid growth anddemanding metabolism1 the result is a comprehensiverewritingupdownregulating certain genes and proteins allowing cells toevade apoptosis and migrate to areas with better oxygenperfusion crucially this microenvironmentally inducedintracellular shift also results in genomic instability gialterations to dna repair and resistance to cell killing bycancer therapies after decades of research it has becomeclear that the relevance of hypoxia for both oncogenesisand treatment resistance is inescapablein the context of radiotherapy rt a link betweenresistance and low intratumoral oxygen pressure has beenknown since the publication of a study modelling oxygenï¬ow in lung tumours years ago2 for some cancerssuch as head and neck squamous cell carcinomashnsccs hypoxia is a major contributing factor to localrt failure3 advancesin developing technologiesallowing for more precise delivery of rtimaging oftumours and sensitisation to treatment while protectingnormal tissues have led to improved locoregional controland quality ofsincelife for patients78 howeverofï¬cial of the cell death differentiation associationtreatment for many cancers like hnsccs which incidentally are also some of the most hypoxic depends onrt the hypoxic problem remains particularly pertinent9in recent years efforts have been made to correct orreverse hypoxia including administering hyperbaric oxygen therapy to patients1011 reducing cellular oxygenconsumption12 and increasing blood vesselfunctionality1314 to more precisely tackle resistance induced byhypoxia researchers have also soughtthethreshold of treatability of hypoxic tumours by use ofsensitizers15 as a third arm in our battle plan researchhas also gone into developing methods to detect hypoxiaincluding the use of specialised imaging techniques petct scanning combined with hypoxiadetecting radionuclides often studied in tandem with genetic signaturesseeking to genotypically deï¬ne these tumours1819to lowerhowever very few of these advancements have allowedus to overcome hypoxiainduced radioresistance rrthough research activity in this area remains strong amore complete understanding of how the hypoxic environment contributes to rr particularly by modulation ofpotentially targetable dna damage response ddrpathways is warranted this review will outline our current knowledge of the molecular processes that underpinhypoxic rr particularly in the reprogramming of the ddrradiotherapy mechanism of actiontherequirement of oxygenseminal work by gray and colleagues during the 1950sproved that the efï¬cacy of rt was dependent on theavailability of oxygen within the tissue22021 radiationinduces damage through the direct and indirect generation of double stranded breaks dsbs in dna in thepresence of oxygen damage induced is times morelikely to end in cell death22 this effect is best explainedby the oxygen fixation hypothesis where radicals produced directly or indirectly by ionizing radiation ir areoxidised to dna in the presence of oxygen23 making thedamage irreversible24 thistothe hypothesis with the notion that these lesions cannotbe restored to an undamaged state as the damage isï¬xed to dna by oxidisation25 thus without oxygendamage induced is transient and hypoxic cells experiencefar reduced radiationassociated damagelast pointis crucialthough crucial the requirement of oxygen to inducedamage is not where the story ends for rr as it does notfully explain the level of rr we observe this is evidencedby the fact that restoration of oxygen to tumours forexample through applying hyperbaric oxygen does notrestore radiosensitivity26 importantlyit also does notaccount for changes that occur with respect to dnarepair which have been shown to be crucial in impactingthe radiation response27 as these changes are retainedpast the point of radiotherapy administration 0cbegg and tavassoli cell death discovery page of the landscape of the hypoxic tumourin vivo the hypoxic region exists on a gradient ofoxygen pressures with oxygen levels throughout the tissue ranging between severe hypoxia mildhypoxia and anoxia with around consideredphysoxia see table for deï¬nitions tissue oxygenpressures are usually measured in mmhg however sincethe majority of research on hypoxia and the ddr hasbeen performed in vitro where oxygen levels are measured in percent for the purpose of this review o2 willbe referred to predominantly the difference betweenin vivo and in vitro measurements of oxygen is importantthough with tissue normoxia physoxia classiï¬ed ataround o2 or mmhg and in vitro normoxia beingaround o2 fig the hypoxic tumour is a space of restricted proliferationparticularly when oxygen levels are o2 cell cyclearrest and decreased protein synthesis juxtaposed againstaccelerated aggressivity microenvironmental interactionsand altered ph28 at the most oxygendepleted borderexists the barren land of necrosis with the highly proliferating and comparatively treatmentsensitive aerobiccells closest to the blood vessel fig tumour hypoxia does not develop in a linear fashionand is highly heterogenous and changeable the hypoxictumour is dynamic with ï¬uctuating vessel functionalityand cycling oxygen levels creating regions of acute andchronic hypoxia31 where part of the tissue may sufferacute hypoxia after temporary occlusion of a blood vesselsocalled perfusion limited in which oxygendeprivationcycles last sometimes minutes sometimes hours beforesubsequent reoxygenation chronic hypoxia is diffusionlimited where oxygen levels become a factor of distancefrom the blood vessel31 compounded with this is thediffering rates of oxygen consumption and responsivenessto oxygen availability in cells within the tissue to be ableto fully understand and ultimately treat the hypoxictumour it must be remembered that the changeability ofoxygen concentrations in the hypoxic tumour also predictably uences its behaviour and response to treatment in the context of radiotherapy cells with o2 levels is where we see most resistance so called radiobiological hypoxia24 whether this is acute and thereforefollowed by reoxygenation or chronic oxygen deprivedfor more than h can have marked differences on theensuing genomic and proteomic changes that ultimatelyallow for hypoxic survival32 thus within one tumourdifferent regions are likely to have a completely differentresponse to the same dose of radiotherapyitaside from the oxygen status the involvement of otherenvironmentalfeatures affected by hypoxia must beconsidered an additional outcome of hypoxic adaption isthe concomitant phenotypic shift of the microenvironmentis now accepted that hypoxia can induceammation33 demonstrated even by patients whodevelop mountain sickness after prolonged periods athigh altitude34 hypoxic tumours are known to havehigher ltration of protumour immune cells such asm2 macrophages35 a feature known to be involved inrr3637 the same could also be said with respect to thehypoxicinduction of highly rt resistant cancer stemcells38 though this subject needs further research thelikelihood of an interplay between intracellular geneticreprogramming as a result of hypoxic adaption and themicroenvironment in mediating radioresponse is stronggenetic reprogrammingthe hifswithin this chaotic showground of heterogeneity andat the core of cellular adaption to hypoxia are the alteredgenetic pathways that push for survival against adversitycommonly dysregulated genes include glut1 involvedin altered glucose metabolism vegf involved intable glossary of terms multiple classiï¬cations of the terms used to describe hypoxia exist throughout the literaturethis represents a general consensus and what is used in this reviewtermhypoxianormoxiaphysoxiaanoxiasevere hypoxiamild hypoxiaacute hypoxiachronic hypoxiadeï¬nitionreduced oxygen levels usually ¤ o2 mmhg in in vitro studiesnormal atmospheric oxygen used in in vitro studies o2 mghgphysiological levels of oxygen in tissues between mmhg tissue speciï¬c see fig complete absence of oxygen o2 o2 o2incubation in hypoxic conditions hincubation in hypoxic conditions hradiobiological hypoxiaoxygen levels where the efï¬cacy of radiotherapy is half maximal mmhg04 o2ofï¬cial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of fig approximate oxygen levels reported in different tissues in mmhg used in in vivo experiments and o2 used in in vitroexperiments note that normal tissue normoxia or physoxia is considerably less than the o2 used in vitro as normoxia adapted frommckeown139 liu140 and graham26fig the heterogeneity of the hypoxic tumour tumours suffer from reduced oxygen availability due to the disanised nature of thevasculature where occlusion of a blood vessel bv occurs tumours are said to be under perfusion limited hypoxia pl hypoxia where lack ofoxygen is a function of distance from the vessel cells experience diffusion limited dl hypoxia when these states are temporary h it is said tobe acute or chronic when h within hypoxia tumour cells undergo considerable genetic reprogramming contributing to therapy resistance andmetastatic behaviourofï¬cial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of neoangiogenesis and lox involved in remodelling ofthe extracellular matrix7in the nobel prize in physiology or medicine wasawarded to three scientists gregg semenza williamkaelin and sir peter ratcliffe for their contributions toour understanding of cellular oxygensensing mechanisms39 this included the discovery of a group of transcription factors regulated by hypoxia that allow forcellular adaption40 these hypoxiainducible factor hifproteins hif13 are transcription factors composed oftwo subunits the α subunits reside in the cytoplasm andto rapid degradation min41 underare subjectnormal circumstances this degradation is mediated bythe actions of prolylhydroxylasesphd14 whichhydroxylate hifα at the oxygendependentdegradationdomains oddd of note phd2 and phd3 are themselves transcriptional targets of hif alluding to possiblenegative feedback systems in place though conï¬ictingresults suggest this system doesnt always function effectively to constrain cancer growth42 subsequentlyhydroxylation by phds recruits the vonhippellandauvhl protein45 this alongside other proteins forms ane3 ubiquitin ligase complex ubiquitinating hifα forproteasomal degradationin hypoxia due to the lack of molecular oxygen neededfor hydroxylation4246 this degradation cascade does nottake place and hifα subunits translocate to the nucleusto associate with hifβ subunits47 the hif complex ininteraction with its coactivators p300 and crebbindingprotein cbp then binds to hypoxia response elementshresin dna to initiate transcription of hiftarget genesintheincludingcontrolledadditional layers of hif regulation also exist to keepthis pathway in check such as factor inhibiting hif fihwhich hydroxylates hif subunits at asparagine residuesblocking their association with p300cbp48 some evidence has shown that hifs also undergo other posttranslational modiï¬cations including phosphorylation andacetylation as a further method of regulation4247 however as with many such processes in cancer it can beaberrantlyhypoxiaindependent stabilisation of hifα by oncogenes such asegfr and mtor4950 and depletion of hifregulatoryfactors4251 the hif proteins themselves can interactwith a number of factors relevant in cancer such as p53mutants present in human papillomavirus hpvnegativehnsccs and nonsmall celllung cancers nsclcsresulting in transcriptional control of protumorigenicgenes52 since both hifs and p53 compete for binding ofp300cbp to enact transcriptional control the hifs havea unique relationship with this highly cancerrelevantprotein53 inactivation of p53s transcriptional abilities hasbeen observed54 though again conï¬icting results exist forthis55ofï¬cial of the cell death differentiation associationmost of the work investigating hifdirected transcriptional changes in hypoxia has focussed on the actions ofthe bestknown hif hif1 however both hif2 andhif3 also play a role in hypoxic transcriptional control42interestingly relative expression of the hifs has beenshown to differ between hypoxic tissues demonstratingthat each may have speciï¬c functions56 in some casesthey may indeed work in concert as hif2 has beenshown to be induced when hif1 is depleted50hifs are master regulators of the hypoxic response andconcurrent with the notion that hypoxic tumours areradioresistant depletion of hif1α in tumour modelsradiosensitises cells41 one study showed that intermittenthypoxia showed less radiationinduced cell death bothin vitro and in mice via stabilisation of hif1α57 thisinvestigation also found that intermittent hypoxia had amore signiï¬cant effect than chronic hypoxia hif1α hasalso been shown to function via the hif1αmyc pathway in which hif1α competes with the transcriptionactivator myc for sp1 binding in the target gene promoter to downregulate mismatch repair mmr genesmsh2 and msh6 in o2however how exactly hifs contribute to rr of thehypoxic tumour be itthrough their transcriptionalfunctions or interactions with other proteins is so farunresolved notably some radio and chemotherapiesthemselves upregulate or stabilise hifs41though genetic reprogramming in hypoxia can lead to anumber of alterations for the purpose of this review wewill focus on those associated with rr and ddr formore general reviews see schito60 and tsai61reprogramming of the ddrthe ddr is a complex process consisting of overlapping and interconnected pathways initiated by differentforms of dna damage arguably one ofthe mostimportant homeostatic processes it allows us to withstand constant and numerous dna damageinducinginsults the result of this protection ensures that onlyreliable genomes are passed on to the next cellular generation for cancer considering both the power ofmutagenesis in driving oncogenic potential and the factthat many cancer therapies function by inducing dnadamage the ddr has considerable relevance for therapyresistance and tumour progressionrepair of dna is a tale of three acts ï¬rstly the damagepropagates a signal that recruits sensors to the site ofdamage secondly the signal is ampliï¬ed by transducersand thirdly response pathways are initiated by effectorsfor each part of the process welldeï¬ned though notexclusive sets of proteins act as sensors transducers andeffectors respectively28 shrouding these repair processesare signals to stall the cell cycle initiated by chk1 orchk2activated cdc25 and p21to allow time for 0cbegg and tavassoli cell death discovery page of clearance of this damage and initiation of apoptoticpathways for example as initiated by atms interactionwith p53 if the repair is unsuccessful6263for the repair of radiationinduced dsbs two primarypathways are put to use homologous recombination hrand nonhomologous end joining nhej the formerconsidered less errorprone uses sister chromatids torepair dna and as such can only take place during g2sphase of the cell cycle nhej predominates in g1 but canoccur at any stage of the cell cycle and often results in thegeneration of insertiondeletion mutations which havethe potential to lead to more oncogenic alterations hr ismediated primarily by the recruitment to sites of damageof master transducer of the dsb response atm ataxiatelangiectasia mutated a phosphoinositide3kinaserelated protein kinase pikk following detection by themrn complex composed of mre11rad50nbs1which initiates activity of effectors including rad51 andbrca1 nhej occurs following sensing of damage by theku proteins ku70 and ku80 and signal transduction ofku in complex with dnapkcs dnadependent proteinkinase catalytic subunittogether forming dnapk andsubsequent activity of effectors dna ligase iv ligivand xrcc464alteration of ddr pathways has been seen across manycancers compared to normal tissue perhaps the mostwellknown are the mutations in brca12 in aggressivehereditary breast and ovarian cancers65 understandablywhere hypoxia represents an exaggerated form ofaggressive tumours the ddr pathways in hypoxia operatedifferently to those in normoxia indeed this is true forevery aspect of the ddr process dna damage in theform of dsbs is reduced in conditions of hypoxia o2and hypoxia alone does not induce dsbs2466 researchhas shown that different members of the ddr pathwayscan be either activated or downregulated in conditions oflow oxygen see tables and for reported alterationsto hr nhej and mismatch repair mmr pathwayscrucially whether the cells are in acute or chronichypoxia or h and at what level of oxygen depletion may deï¬ne the ensuing response despite this delineation there lacks within the literature proper reportingof experiments carried out in either acute or chronic mildor severe hypoxia with interchangeability in use of termssee table for a consensus of parameters used with thesedeï¬nitionssensorsthe mre11rad50nbs1 mrn complex is responsible for sensing dna damage and initiating both the hrand nhej pathways by recruitment of transducers such asatm via nbs167 while the mrn complex is consideredthe main sensor responsible for recruiting and activatingatm followingdamage atrip atrinteractingofï¬cial of the cell death differentiation associationprotein and ku7080 are sensorsresponsible forrecruitment of atr and dnapkcs respectively67 fig though there are many overlapping interactions atminatminteracting protein with roles in replication stressrs genome stability and the base excision repair berpathway has also been shown to recruit atm independent of dna damage68repression of the mrn machinery has been seen inchronic hypoxia days in a medulloblastoma modelwith transcriptomic downregulation of both mre11a andnbs1 resulting in downregulation of etoposideinducedatm and p5369 nbs1 has also been shown to stabilisehif1α particularly in response to ir70 while hif1α hasbeen shown to downregulate nbs1 the authors of onestudy where reduction of nbs1 was seen after h in o2 noted that this repression resulted in the induction ofγh2ax and 53bp1 foci in hypoxia suggesting the presence of dna breaks59 interestingly all components ofthe mrn were found to be downregulated both at themrna and protein level in nsclcs harbouring egfrmutations incubated in severe hypoxia o2 thisdownregulation in egfrmutated cells correlated withtheir increased sensitivity to egfrinhibiting drugs71sensing of damaged dna is a crucial step in theinitiation of repair and begins with changes to the chromatin72 γh2ax a phosphorylated variant of histoneh2ax is induced by mrn activation and accumulates atsites of damage in the chromatin preceding recruitmentand necessary for retention of key ddr signalling proteinsincluding mrn and atm73 studies also show thatγh2ax is crucial for retaining mediators such as 53bp1p53 binding protein mdc1 mediator of dnadamage checkpoint and brca1 at sites of damage66h2ax is primarily phosphorylated by atm but can alsobe phosphorylated by atr and dnapkcs63 indeed aswell as by radiation and chemotherapies γh2ax has alsobeen shown to be induced by hypoxia following replication fork stalling this phosphorylation has been shown inchronic severe hypoxia to occur in a hif atr or atmdependent manner74 crucially some evidence hasshown the phosphorylation of h2ax present only inproliferating cells7778 the downstream effects of thisactivation have been linked to other consequences ofhypoxic regulation including angiongenesis79 via induction of vegf80 experimentally resolution of γh2ax fociafter irradiation is often used as a marker of dsb repair astheory dictates that the phosphorylation should disappearafter damage is repaired however in hypoxia this heavilyrelied upon protocol may necessitate further ï¬netuningku70 and ku80 together forming a heterodimericcomplex tether damaged dna at breaks and are keysensors of dsbs responsible for recruitment of dnapkcs as part of the nhej pathway63 the ku complex hasbeen shown to be both upregulated and downregulated by 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the homologoursrecombination hr pathways in hypoxiaprotein role in ddrmechanism of alterationalteration conditions and consequencesreferencenbs1sensor of dsbs in hr activated atmas part of the mrn complexcid129 pasb domain of hif1αmre11sensor of dsbs in hr activated atmas part of the mrn complexcid129 atmtransducer of hr in dsb repaircid129 autophosphorylation atser1981atrtransducer of dna repair induced byreplication stresscid129 rad51effector of dsb repair in hrrad52effector of dsb repair in hrrad54 motor protein effector of dsbrepair in hrbrca1effector of dsb repair in hrcid129 e2f4p130cid129 lsd1cid129 ezh2cid129 mir210cid129 mir373cid129 mir210cid129 cid129 e2f4p130cid129 h3k4 demethylation via lsd1cid129 downregulated in chronic mild hypoxia days o2cid129 downregulation in acute mild h o2cid129 resulted in induction of γh2ax and 53bp1 focicid129 downregulated in chronic mild hypoxia days o2cid129 activated in acute hypoxia o2cid129 increased expression and activity o2 hcid129 mediated by src and ampk signallingcid129 activated in acute o2cid129 resulted in phosphorylated p53 andaccumulation and growth arrestcid129 downregulation in chronic severe hypoxia o2 h and or hcid129 decreased radioresistancecid129 increased genomic instabilitycid129 downregulation in o2 hcid129 decreased mrna expression o2 hcid129 downregulated o2 hcid129 decreased mrna expression o2 hcid129 downregulation in chronic severe hypoxia o2 hcid129 decreased mrna expression o2 hcid129 downregulation in o2 hcid129 decreased radioresistancecid129 decreased expression o2 hcowman69to59cowman69hashimoto88bencokova28hammond75meng119bindra oliveira82meng119crosby118meng119meng119lu117bindra120oliveira82meng119brca2effector of dsb repair in hrcid129 hypoxia in different studies81 one study found downregulation of ku80 after h in mild hypoxia o282another using severe hypoxia o2 found upregulation of ku70ku80 in a431 cells alongside many othermembers of the nhej pathway and proteins generallyinvolved in metastatic progression83 a study in humanand mouse hepatoma cells found upregulation of the kuheterodimer upon incubation in hypoxia o2 or withhypoxia mimics and downregulation associated with hif1βdeï¬cient cells84 alternative subpathways of nhejalso exist possibly as insurance for when classical nhejmediators are inoperative howeverthe impact ofhypoxia on these pathways has not been extensivelystudiedofï¬cial of the cell death differentiation associationtransducersatm and atr are two of the most important proteinsinvolved in transduction of the ddr as pikk familymembers they phosphorylate a number of proteinsinvolved in propagating the signal and repairing dna aspart of both the hr and nhej pathways as well asundergoingtheresponse until dna is repairedautophosphorylationto maintainbroadly speaking atm has been shown to be activatedparticularly in acute hypoxia as shown in the study bybencokova et al the pattern of activation in this contextdoes not match rtinduced atm activation which follows mrn recruitment to dsbs85 as atm phosphorylation does not correlate with the presence of dsbs 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the nonhomologous endjoining nhej pathways in hypoxiaproteinrole in ddrmechanism of alterationalteration conditions and consequencesreferenceku70ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku70cid129 cid129 dnapkcstransducer of nhej pathwaycid129 autophosphorylation atser2056dna ligiv effector of nhej repairxrcc4effector of nhej repaircid129 cid129 cid129 decreased mrna expression o2 hcid129 upregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 hcid129 upregulation o2 hcid129 downregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 hcid129 decreased mrna expression o2 hcid129 increased expression and activity o2 hcid129 activated in mild hypoxia o2 led to positiveregulation of hif1 and upregulation of glut1cid129 decreased mrna expression o2 hcid129 decreased mrna expression o2 hmeng119ren83lara81um84oliveira82ren83lara81um84meng119hashimoto88bouquet103meng119meng119table a nonexhaustive list showing alterations to sensors transducers and effectors of the mismatch repair pathwayin hypoxiaprotein role in ddrmechanism of alterationmlh1dimerises to pms2 to form themutlα complex in mmrpms2dimerises to mlh1 to form themutlα complex in mmrmsh2dimerises with msh6 forms themutsα complex in mmrmsh6dimerises with msh6 forms themutsα complex in mmrcid129 mad1maxcid129 mntmaxcid129 dec12cid129 mir155cid129 lsd1cid129 hdaccid129 hypoacetylationhypermethylation on h3cid129 cid129 mycmaxcid129 hif1α via sp1cid129 mir155cid129 hcid129 p53cid129 hif1α via sp1cid129 mir155cid129 hdaccid129 p53cid129 hypoacetylationhypermethylation on h3ofï¬cial of the cell death differentiation associationalteration conditions andconsequencescid129 downregulation h o2cid129 downregulation in h o2cid129 increased expression h o2resulting in genomic instability instem cellscid129 downregulation at protein level h o2cid129 resulting in genomic instability instem cellscid129 downregulation h o2referencebindra121127mihaylova128nakamura129rodriguezjimenez145lu115mihaylova128rodriguezjimenez145bindra121127koshiji58cid129 downregulation h o2cid129 increased expression h o2resulting in genomic instability instem cellskoshiji58rodriguezjimenez145 0cbegg and tavassoli cell death discovery page of often reduced or absent in severe hypxoia28 the authorsof this study emphasized that atm activation was speciï¬cto the level of hypoxia only phosphorylated at o2and hifindependent since phosphorylation was maintained even in hifknockout cells activation of atmwas attributed to autophosphorylation the result of whichwas an activation of targets much like dna damageinduced atm activation but dependent on the activityof cell cycle regulator mdc128 this study did not analyserr but the results suggest that atm activation byhypoxia is likely enacted as a means of halting the cellcycle in order to allow for dna repairthe pattern of atm activation in hypoxia however isnot clearcut and may depend on cancer type atm canbe regulated by a number of factors as part of the hypoxicresponse including mrn or atmin but also by posttranslational and epigenetic factors86 one study foundthat atm was downregulated along with hif1α by amicrorna mir18 resulting in radiosensitivity87the study by hashimoto et al88 showed atm activation alongside activation of a number of other key ddr orcancerrelated proteins including dnapkcs akt andegfr and decreased expres | 0 |
coronavirus disease COVID19 pandemic access to surgical care for patients with head and neck cancer HNC is limited and unpredictable Determining which patients should be prioritized is inherently subjective and difficult to assess The authors have proposed an algorithm to fairly and consistently triage patients and mitigate the risk of adverse outcomes METHODS Two separate expert panels a consensus panel participants and a validation panel participants were constructed among international HNC surgeons Using a modified Delphi process and RAND CorporationUniversity of California at Los Angeles methodology with consensus rounds and meetings groupings of highpriority intermediatepriority and lowpriority indications for surgery were established and subdivided A pointbased scoring algorithm was developed the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN Agreement was measured during consensus and for algorithm scoring using the Krippendorff alpha Rankings from the algorithm were compared with expert rankings of case vignettes using the Spearman rank correlation coefficient RESULTS A total of indications for surgical priority were rated Weights for each indication ranged from to scale range to The response rate for the validation exercise was The SPARTANHN demonstrated excellent agreement and correlation with expert rankings Krippendorff alpha [ CI ] and rho [ CI ] S The SPARTANHN surgical prioritization algorithm consistently stratifies patients requiring HNC surgical care in the COVID19 era Formal evaluation and implementation are required Cancer American Cancer Society LAY SUMMARY ¢ Many countries have enacted strict rules regarding the use of hospital resources during the coronavirus disease COVID19 pandemic Facing delays in surgery patients may experience worse functional outcomes stage migration and eventual inoperability¢ Treatment prioritization tools have shown benefit in helping to triage patients equitably with minimal provider cognitive burden¢ The current study sought to develop what to the authors knowledge is the first cancerspecific surgical prioritization tool for use in the COVID19 era the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN This algorithm consistently stratifies patients requiring head and neck cancer surgery in the COVID19 era and provides evidence for the initial uptake of the SPARTANHN KEYWORDS coronavirus disease COVID19 delivery of health care head and neck cancer health priorities patient selection surgical procedures waiting listsCorresponding Author John R de Almeida MD MSc Division of Surgical Oncology Department of OtolaryngologyHead and Neck Surgery 8NU883 Toronto General Hospital University Health Network Elizabeth St Toronto ON M5G 2C4 Canada Johndealmeidauhnca Division of Surgical Oncology Department of OtolaryngologyHead and Neck Surgery Princess Margaret Cancer Center University Health Network University of Toronto Toronto Ontario Canada Institute of Health Policy Management and Evaluation Dalla Lana School of Public Health University of Toronto Toronto Ontario Canada Division of OtolaryngologyHead and Neck Surgery Dalhousie University Halifax Nova Scotia Canada Division of OtolaryngologyHead and Neck Surgery McMaster University Hamilton Ontario Canada Department of OtolaryngologyHead and Neck Surgery Western University London Ontario Canada Department of OtolaryngologyHead and Neck Surgery Memorial Sloan Kettering Cancer Center New York New York Head and NeckEndocrine Oncology Moffitt Cancer Center Tampa Florida Department of OtolaryngologyHead and Neck Surgery Medical University of South Carolina Charleston South Carolina Department of OtolaryngologyHead and Neck Surgery Stanford University Palo Alto California Department of OtolaryngologyHead and Neck Surgery University of Michigan Ann Arbor Michigan Department of OtolaryngologyHead and Neck Surgery The University of Texas MD Anderson Cancer Center Houston Texas Head and Neck Unit The Royal Marsden Hospital London United Kingdom Department of OtolaryngologyHead and Neck Surgery Icahn School of Medicine at Mount Sinai New York New York Department of OtolaryngologyHead and Neck Surgery Sunnybrook Health Sciences Centre University of Toronto Toronto Ontario Canada Department of OtolaryngologyHead and Neck Surgery Sinai Health System University of Toronto Toronto Ontario CanadaThe first authors contributed equally to this Additional supporting information may be found in the online version of this 101002cncr33114 Received June Revised June Accepted June Published online Month in Wiley Online Library wileyonlinelibrarycomCancer Month 0cOriginal INTRODUCTIONOn March the World Health anization declared a global pandemic due to the novel coronavirus severe acute respiratory syndrome coronavirus SARSCoV2 and the resulting coronavirus disease COVID191 As a result in many jurisdictions operating room capacity has been limited to only emergent or urgent surgical procedures2 Several advisory bodies have issued recommendations to safeguard access to oncologic surgery while still acknowledging that treatment delays may be necessary The American College of Surgeons has recommended postponing elective surgery including for patients with lowrisk cancers while recommending that other urgent cancer surgeries proceed34 Cancer Care Ontario has issued similar guidance recommending that hospitals include cancer surgery in their care delivery plan5The time from the diagnosis of head and neck cancer HNC to surgery is a metric with prognostic importance with treatment delays portending poorer oncologic outcomes68 In a recent systematic review evaluating delays in time from diagnosis to treatment initiation of studies demonstrated a decrease in survival to be associated with treatment delays68 These data support the urgency of initiating treatment for patients with HNC but to our knowledge do not inform a stratification schema when operating room access is not available for all patientsAs a result of these new imposed constraints difficult decisions regarding prioritization for cancer surgery are obligatory and require the consideration of broader principles regarding scarce resource allocation9 Key among these is the need for consistency and transparency to achieve fairness and to avoid engendering disparities in both access and outcomes1011 Prioritization on a casebycase basis using expert clinical judgment can be logistically challenging carries a cognitive burden and is susceptible to the biases of practitionersSurgical prioritization tools or algorithms offer decisionmaking transparency and provide equitable and timesensitive access to care to the patients who need it most1213 Although tools for surgical prioritization in the era of COVID19 continue to emerge to our knowledge oncology patients have not been explicitly considered14 Herein we have presented the development and validation of a novel algorithm Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN for the prioritization of surgery for patients with HNCMATERIALS AND METHODSThe current study was granted a waiver from the research ethics board at the University Health NetworkParticipants and SettingFor instrument development a group of expert HNC surgeons JRD DPG RG JCI DBC DB AE DJE KMH EM and IJW from institutions University Health Network Sinai Health Systems and Sunnybrook Health Sciences Centre at the University of Toronto participated in the consensus process consensus panel At the time of the consensus process all institutions were operating under significant resource constraints with limited availability of operating room time For instrument validation a group of participants JRD CWN DF DPG and EM completed the scoring algorithm designed after the consensus process Fifteen external head and neck surgeons HZ ACN RJW MAC CM EMG VD AGS AJR CML EYH JM VP BM and EG from institutions across Canada institutions the United States institutions and the United Kingdom institution participated in a ranking exercise of clinical vignettes validation panelScopeThe scope of variables considered in the prioritization algorithm was established and vetted by the consensus panel see Supporting Information All indications for prioritization were presented to the consensus panel using an online survey platform Google Forms httpsdocsgooglecomforms With exceptions survey respondents were asked to consider each of the indications in isolation For wait times panel members were asked to also consider histologic grade Similarly for surgical site the panel was asked to simultaneously consider extent of surgery Related indications were presented sequentially to facilitate pairwise comparison eg stage I and II vs stage III and IV were presented in sequence AJCC 8th edition The list of indications was pilot tested by surgeons JRD DPG EM and RG for sensibility readability content validity language and comprehensibilityConsensus ProcessThe consensus panel participated in a Delphi consensus process with rounds of rating see Supporting Information The first rounds aimed to achieve consensus regarding the priority grouping high intermediate or low High priority was defined as an indication to Cancer Month 0cproceed to surgery within weeks The second rounds of rating involved ranking each indication less important neutral or more important within their respective priority grouping Two teleconference meetings were conducted between the first and second rounds and between the third and fourth rounds with anonymized results from the prior round presented for discussion and to address inconsistencies and misinterpretationsA modification of the RANDUniversity of California at Los Angeles UCLA method was used to achieve consensus15 This methodology typically is used to determine the appropriateness of an intervention but in this setting was used to determine surgical priority We used a scale ranging from to in rounds and to indicate the decision to not operate or low priority scores intermediate priority scores or high priority scores For rounds and we used a scale from to to rate each indication compared with other indications within each of the priority groupings as either less important neutral or more important Consensus was determined based on RANDUCLA criteria15 For the first rounds to determine surgical priority a hierarchical logic was adopted to determine consensus regarding whether surgery should be performed and to then determine the priority of surgery based on the given indication Agreement on the decision to not operate was defined as a minimum of of the panelists rating a given indication with a zero score If there was no agreement to avoid surgery agreement for surgical priority then was defined as ¤ panelists rating the indication outside the 3point range containing the median as per RANDUCLA guidelines15 For rounds and any indication that failed to achieve consensus was classified as being of intermediate priority and for rounds and any indication failing to achieve consensus was classified as neutral within the priority groupingDevelopment of the SPARTANHNThe algorithm uses a pointbased system to assign a total score based on the sum of the individual indication scores see Supporting Information with higher scores corresponding to higher priority Scoring weights were based on consensus from both sets of rounds such that highpriority indications were assigned scores ranging from to intermediatepriority indications were assigned scores ranging from to and lowpriority indications were assigned scores ranging from to Within each priority grouping 3point range the scores were assigned based on the consensus ratings from the third and fourth rounds For any patients with the same total score the SPARTANHNde Almeida et alpatient with the longer surgical wait time was assigned the higher priority rankClinical VignettesTwelve clinical vignettes were constructed see Supporting Information after the consensus rounds to validate the SPARTANHN The vignettes described a variety of clinical scenarios incorporating multiple prioritization indications and additional clinical information Experts were asked to consider only the patientlevel information provided to them and not their own unique clinical and community practice environments Twelve scenarios were selected for diversity of cases The number was considered appropriate while avoiding the excessive cognitive burden associated with ranking too many scenariosStatistical AnalysisAgreementAgreement between raters during the Delphi process was calculated at each round and within each priority grouping using the Krippendorff alpha Kalpha Because typical coefficients of reliability are not suitable for coded data agreement for the rank orders generated by coders JRD CWN DF EM and DPG applying the SPARTANHN algorithm to the clinical vignettes was assessed using Kalpha calculated with bootstrap samples16 The Kalpha allows for estimation of reliability for any number of raters and categories and may be used when there are missing data17Validity of the SPARTANHN AlgorithmConvergent validity of the median rankings from the coders of each of the vignettes using the SPARTANHN and the expert panel rankings were assessed using the Spearman rank correlation coefficient The strength of the correlation was considered weak if the rho was moderate if the rho was between and and strong if the rho was In addition to SPARTANHN a second algorithm using a decisionmaking flowchart was developed SPARTANHN2 The tool and associated performance characteristics are included in Supporting Information Sample Size ConsiderationsFor determination of an adequate sample size for the expert panel we assumed that for model validity there was a strong correlation between the model rank order and expert rank order ie rho ¥ an alpha of power of and a nonresponse rate of Therefore the calculated sample size requirement was participantsCancer Month 0cOriginal All analyses were 2sided and statistical significance was set at P\xa0¤\xa0 Analyses were conducted using SAS University Edition statistical software SAS Institute Inc Cary North CarolinaRESULTSEstablishing Consensus Priority Groupings First Consensus RoundsAfter the first rounds the panel failed to achieve consensus for any indications that would result in a decision to not operate More than respondents indicated that they would not operate for the following indications the availability of alternative nonsurgical treatment with a similar prognosis respondents poor performance status ie Eastern Cooperative Oncology Group [ECOG] performance status of respondents and very severe comorbidity as indicated by a noncancerspecific survival rate of at year respondents In the first round consensus was achieved for indications for surgical prioritization of which were considered high priority of which were considered intermediate priority and of which were considered low priority After review of firstround results consensus was achieved for an additional indications indications were rated as being of intermediate priority and indications were rated as low priority Table Establishing Ranking Within Each Priority Grouping Second Consensus RoundsOf the lowpriority indications consensus for the importance of factors was achieved for scenarios both of which were deemed less important Table Of the intermediatepriority indications consensus for the importance of factors was achieved for of scenarios Of highpriority factors consensus for the importance of factors was achieved for scenarios all of which were deemed to be more importantAgreement during consensus rounds was found to be weak to moderate for all rounds ranging from to The agreement was similar when measured as per priority grouping in which the Kalpha ranged from to Table SPARTANHN Surgical Prioritization Scoring SystemPriority weights for each indication ranged from to spanning a 9point range and translated from the rounds of priority groupings into categories Four indications were assigned a weight of based on consensus that these factors were both high priority and more important Supporting Information Table All other highpriority indications were assigned a weighted score because there was no consensus that they were either less or more important For intermediatepriority indications a weighted score of was assigned for of the indications deemed to be more important by consensus The other indication deemed to be more important thyroid cancer with tracheal invasion was assigned a score of because of the fact that this indication can be associated with lowgrade histology which is assigned a negative weighted score Three intermediatepriority indications that were rated as more important were resource use indications which generally are colinear As such the decision was made to assign a maximum score of for the presence of any or all of these indications One intermediatepriority indication was deemed to be less important by consensus and was assigned a score of All other intermediatepriority indications were assigned scores of For the lowpriority indications those deemed to be less important were assigned a weight of and all other indications were assigned a weight of The total scale score ranged from to Fig Reliability and Validity AssessmentAgreement between the coders for the SPARTANHN was excellent Kalpha Agreement between the expert raters was moderate Kalpha Convergent validity was demonstrated by a strong correlation between the rank orders generated by the SPARTANHN and external experts rho CI [P\xa0 a0 ] Agreement between expert rankings and SPARTANHN rankings for the vignettes is shown in Figure DISCUSSIONIn the setting of the COVID19 pandemic in which the availability of operating room time as well as hospital and intensive care unit beds is limited the prioritization of surgical oncology cases is imperative to mitigate downstream adverse outcomes1920 The current methodology was adopted based on expert consensus In the current study we have proposed the SPARTANHN with the objective of providing transparency and facilitating surgical prioritization for treatment providersCreating COVID19era allocation schemas that are ethically sound is both critical and challenging Emanuel et al have advocated ethical principles with which to Cancer Month 0cSPARTANHNde Almeida et ali ytidbrom lanoitcnufi tnacifings laitnetoP fi ytilibareponi rohtw romut fi tnemriapmi citem etaredom laitnetoPsoc ro lanoitcnuf htiw recnac doryhTiinosavni laehcarthtw romut inossergorp esaeisd citamotpmySENEtsil tiawno e lihwTR suoverPi dedeecxe emit tiaW dedeecxe emit tiaWihgh rof kw¥ yb liygootsh edargihgh rof kw yb liygootsh edarg igngami ro lacniilC hpmyl decnavdAegats gncnavdai ei inossergorpi cpocsorcam roN ge esaesd edoni esaesdV inoitceserI ot III egatSnoitceser enob htiw recnac ytivac larOnoitide ht CCJA yregrus fo htgneL latot ro latotraen gniriuqer recnac ytivac larO yats fo htgnel latipsoHh tinu erac evsnetni ioNtinu nwodpets rod yregrus larosnart htiw recnac laegnyrahporOymotcessogllymotoubdnam htiiw recnac laegnyrahporO ymotcegnyral latot htiw recnac laegnyrahpopyHymotcegnyral latot gniriuqer recnac laegnyraLi cpocsodne gniriuqer recnac laegnyrahposaNymotcegnyrahp laitrapdna odne gniriuqer recnac suns lasanarap roi lasaNymotolli xamgniriuqer recnac laegnyrahposaNnoitceser cpocsinoitceseri noitceser nks gniriuqer recnac nks decnavdAinoitcurtsnocer palf lanoger dnai edon hpmyl detimil htiw renac kcen dna daeH edon hpmyl on htiw recnac kcen dna daeHycnangilam enob laropmeTesaesdiII ot I egatSy egAy egAy egAesaesdi SPGOCE y ¥ egAycnangil amditorap edarghgHinoitcurtsnocer palfeerf gniriuqer recnac nks decnavdAi laitrapgniriuqeryregrus laegnyral recnac laegnyraL gniriuqer recnac laegnyrahpopyHnyrahpognyral latotymotcegriuqer recnac sunsi roiretna nepogn i lasanarap ro lasaN ¥i lacgrus lacafonarciili ygootsh edargwol rof dedeecxe emit tiaWkwnoitceser eussittfos htiw recnac ytivac larOi rof gnhcaorppa tubdedeecxe ton em it tiaW dedeecxe emit tiaWliygootsh edarghghiwoliygootsh edargl rof kw deecxe ton emit tiaWromoc ereves yreV SP GOCE ei sutats ecnamrofrep rooP ypareht evitanretlAlebaliava ni dehcaorppa tub edargwol rof kw ditorap edargwoLycnangilam edon hpmyl htiw recnac doryhTiesaesdilygootshi recnacnonge ytidb i ta i lavvrusy srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLi gnknaR fo sdnuoR retfA serocSdna snoitacdn iI noitazitiroirP ELBATCancer Month 0cOriginal TRj tnavuda dna esaesddecnavda hti iw tneitaPderiuqer tinu nwodpets ro tinu erac evsnetniIderiuqer ebut ymotsoehcart oNderiuqer palf eerf oNd yats fo htgnel latipsoHh yregrus fo htgneLd yats fo htgnel latipsoHderiuqer palf eerFh yregrus fo htgneLnoitpo na si SPGOCEsrotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLdeunitnoC ELBATyparehtodar iTRi nosnetxe ladonartxe ENE sutats ecnamrofreppu ygoocnO evitarepooCnretsaE l SPGOCE snoitaverbbAiTABLE Agreement Between Experts During the Delphi ProcessRoundOrdinal ScaleaLCL UCLPer Priority GroupLCL UCLAbbreviations LCL lower confidence limit UCL upper confidence limitThere were raters and agreement was measured using the Krippendorff alphaaOrdinal scale refers to the scale of to used to rate priority of surgery and Per Priority Group refers to the lowpriority mediumpriority and highpriority groups related to the scoring scaleguide the allocation of scarce resources maximizing the benefits produced by scarce resources treating people equally promoting and rewarding instrumental value and giving priority to those patients who are worst off9 These have been contextualized for cancer care more broadly and are manifest in the SPARTANHN algorithm21 The highpriority indications implicitly embrace an underlying premise of saving the most lives andor preserving the most lifeyears Many procedures for patients with HNC are aerosolgenerating and increase the risk to health care workers and other hospitalized patients22 Our process accounted for these by giving consideration to these factors during the consensus process although indications associated with potential exposure to health care workers did not emerge as lowpriority ones Indications associated with lower resource use did achieve consensus for higher importance This may help to avoid the opportunity cost of treating fewer patients with longer surgeriesAnecdotal and institutionspecific prioritization schemas for patients with HNC and general otolaryngology have been suggested213 These parallel similar efforts for general surgery cardiac surgery and orthopedic surgery12132328 In many of these patients are prioritized by the scoring of several criteria and summing of the scores to achieve a total patient score Many of these systems have been validated against expert rankings of surgical priority2728We used a methodology for developing a pointbased prioritization system similar to those previously described29 To the best of our knowledge pointbased surgical prioritization systems have been very well studied to date Hansen et al previously proposed a methodology for developing a pointbased prioritization system using the following steps ranking patient case vignettes Cancer Month 0cSPARTANHNde Almeida et alFIGURE The Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN scoring system ECOG indicates Eastern Cooperative Oncology Group ENE extranodal extensionusing clinical judgment drafting the criteria and categories within each criteria pretesting the criteria and categories consulting with patient groups and other clinicians determining point values for criteria and categories checking the testretest reliability and face validity and revising the points system as new evidence emerges29 Our approach to the development of the SPARTANHN was similar However given the relatively expedited nature of the process we did not directly involve patientsOne method proposed for establishing the priority of all indications in a pointbased scoring system is known as Potentially All Pairwise Rankings of all Alternatives PAPRIKA30 In the current study we chose to use the RANDUCLA process instead of pairwise comparison to minimize computational burden We established Cancer Month 0cOriginal FIGURE External validation rank results A total of experts were asked to rate the scenarios provided shown on the xaxis and the results were compared with the rank generated by models and shown on the yaxis Green shading reflects high priority ranked yellow shading indicates medium priority ranked and red shading indicates low priority ranked Asterisk denotes ties from the algorithm SPARTANHN indicates Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancerindications for surgical prioritization that would create an enormous computational burden using pairwise comparison methodology One problem inherent in the PAPRIKA methodology is the assumption that all indications are not equal and can be ranked However clinically certain indications may be equivalent in priority Furthermore pairwise comparisons assume mutual exclusivity of each of the indications which is not always the case Use of the RANDUCLA consensus process avoids the need for multiple pairwise comparison and allows for consideration of each factor in isolation The goal of the consensus rounds was not to establish a rank order for all indications but mainly to understand which indications result in high intermediate or low priorityThe SPARTANHN algorithm has demonstrated preliminary reliability and validity We demonstrated good agreement between raters and the SPARTANHN algorithm suggesting minimal interpretive error Many of the highpriority indications accounted for some component of interpretation because raters were forced to consider imminent disease progression that may result in an adverse outcome Despite the subjective decisions that must be made as part of SPARTANHN agreement remained high In fact true interrater reliability was found to be higher because the Kalpha is a conservative measure of reliability Other measures of reliability such as the Kendall coefficient of concordance tend to overestimate reliability and cannot be applied to missing data31 Perhaps most important the SPARTANHN correlated highly with expert rankings With established validity this algorithm may be ready for preliminary clinical use although further testing against realworld data to validate it with other cancer outcomes such as survival is neededThe results of the current study must be interpreted within the context of the study design Although externally validated by other surgeons across North America and the United Kingdom the criteria for which consensus was achieved for prioritization were not vetted by patients advocacy groups or other stakeholders such as medical or radiation oncologists The latter groups represent essential providers in the multidisciplinary care of patients with HNC and may have important insight into the availability and effectiveness of nonsurgical treatments1920 Nonetheless the actual prioritization of surgical waitlists remains the sole responsibility of surgeons and their practice partners In addition the SPARTANHN algorithm is intended to assist in making difficult prioritization decisions and is not intended to make recommendations for the time frame in which patients should receive treatment Instead established guidelines should be adhered to for treatment targets Patient wait times as they relate to those targets should be considered when using the SPARTANHN algorithm The validation process in the current study used expert opinion as the gold standard of prioritization which is potentially biased and reflected the opinions of surgeons practicing in academic medical centers from resourcerich nations Subsequently use of the SPARTANHN algorithm in other geographic regions Cancer Month 0cand health care systems requires additional investigation because local treatment paradigms and risk factors may vary substantiallyThe current study has presented the development and validation of a novel algorithm for the prioritization of surgery for patients with HNC Further evaluation of its implementation in various practice settings will be obligatory However the results of the current study have provided data with which to inform realworld use as the current pandemic has obviated our ability to more rigorously study the instrument prior to making necessary and difficult realtime allocation decisionsFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESEvan M Graboyes has received grants from the National Cancer Institute and the Doris Duke Charitable Foundation for work performed outside of the current study Vinidh Paleri offers his services as a proctor for a transoral robotic surgery proctoring program run by Intuitive Surgical and has been remunerated for his time Antoine Eskander has received a research grant from Merck and acted as a paid consultant for BristolMyers Squibb for work performed outside of the current study Ian J Witterick has stock in Proteocyte Diagnostics Inc and has received honoraria from Sanofi Genzyme and Medtronic Canada for work performed outside of the current study The other authors made no disclosuresAUTHOR CONTRIBUTIONSJohn R de Almeida Study idea and design writing and editing Christopher W Noel Study design writing data analysis and editing David Forner Study design writing data analysis and editing Han Zhang Data acquisition and editing Anthony C Nichols Data acquisition and editing Marc A Cohen Data acquisition and editing Richard J Wong Data acquisition and editing Caitlin McMullen Data acquisition and editing Evan M Graboyes Data acquisition and editing Vasu Divi Data acquisition and editing Andrew G Shuman Writing data acquisition and editing Andrew J Rosko Data acquisition and editing Carol M Lewis Data acquisition and editing Ehab Y Hanna Data acquisition and editing Jeffrey Myers Data acquisition and editing Vinidh Paleri Data acquisition and editing Brett Miles Data acquisition and editing Eric Genden Data acquisition and editing Antoine Eskander Data acquisition and editing Danny J Enepekides Data acquisition and editing Kevin M Higgins Data acquisition and editing Dale Brown Data acquisition and editing Douglas B Chepeha Data acquisition and editing Ian J Witterick Data acquisition | 2 |
LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR OBrien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree 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Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary 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Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL OHagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c' | 2 |
Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso and Lucia Altucci Department of Precision Medicine University of Campania Luigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespeciï¬cdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneï¬cial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientiï¬c interest focusing on theidentiï¬cation of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol ï¬setin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modiï¬cations are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modiï¬cations Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT are enzymes classiï¬ed as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identiï¬ed inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespeciï¬c degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair OCallaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context speciï¬city BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deï¬ciency DAngelo Bioactive compounds in the diet can act as antioxidantand antiammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and ï¬setin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health beneï¬ts Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneï¬cial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneï¬cial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and antiammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin ï¬setin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classiï¬cation of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid proï¬le antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidproï¬le antioxidant antiammatoryAntioxidant antiammatoryAnticancer antioxidantantiammatoryAnticancercardiovascularpreventive antiammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesDAngelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonï¬avonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has antiammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger antiammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits proammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modiï¬es SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneï¬cial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneï¬cial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneï¬cial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress ï¬brosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together theseï¬ndings highlight the beneï¬cial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneï¬cialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been conï¬rmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective beneï¬t through improvement ofendothelial functionammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well deï¬ned Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientiï¬c research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efï¬cacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimers disease Parkinsonsdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood ï¬ow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneï¬cial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe ï¬avonoid polyphenol quercetin Que ²²pentahydroxyï¬avone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and antiammatory properties Nabavi Wu In recent years the scientiï¬c community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inï¬ammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inï¬ammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inï¬ammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the bodysantioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseasesammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable conï¬guration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cells antioxidant defense mechanisms moderateoxidative stress can in fact increase the cells antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These ï¬ndings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientiï¬c community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere signiï¬cantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpeciï¬cally in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats conï¬rming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these ï¬ndings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneï¬cial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ammatoryconditions is reported in Table Speciï¬cally a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and antiammatory capacities this ï¬avonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary ï¬brosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclariï¬cation Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneï¬cial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer antiammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneï¬cial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInï¬ammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efï¬cacy of Provex CV Supplement toReduce Inï¬ammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an antiammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a signiï¬cant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These ï¬ndings were also conï¬rmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Speciï¬cally BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and antiammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ammation signaling Speciï¬cally BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is | 2 |
craigpattersontelecaretoj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per loncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatientssaï¬f al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset ogorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseandimplicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 commonduringadversetheoutbreakconï¬ict of interest the authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | 0 |
"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 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functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu | 0 |
netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimers and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth peoples hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term netosisshould only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3 levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimers disease alzheimers disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimers disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patients ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohns disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohns patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csas efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation httpsdoi101038s414180170012 gupta s and mj kaplan the role 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Pathwayspecific model estimation for improved pathway annotation by network crosstalkMiguel CastresanaAguirre Erik L L SonnhammerPathway enrichment analysis is the most common approach for understanding which biological processes are affected by altered gene activities under specific conditions However it has been challenging to find a method that efficiently avoids false positives while keeping a high sensitivity We here present a new networkbased method ANUBIX based on sampling random gene sets against intact pathway Benchmarking shows that ANUBIX is considerably more accurate than previous network crosstalk based methods which have the drawback of modelling pathways as random gene sets We demonstrate that ANUBIX does not have a bias for finding certain pathways which previous methods do and show that ANUBIX finds biologically relevant pathways that are missed by other methodsImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisThere are four generations of pathway enrichment analysis approaches Overrepresentation analysis ORA calculates how many genes from a list of genes extracted based on a threshold or criteria eg differentially expressed genes are in a certain pathway1 Statistical significance is assessed repeating this process with a background list of genes eg all the genes in the microarray This is known as Gene Enrichment Analysis GEA and famous tools like DAVID2 use it Similar but taking into account all the genes in the experiment and the gene expression values is the Functional Class Scoring algorithms FCS3 for which known algorithms include Gene Set AnalysisGSA4 and Gene Set Enrichment Analysis GSEA5 However both FCS and ORA have limitations They both consider genes as independent which is often not true only taking into account their overlap and not their associations or interactions6 Another issue with overlapbased methods is their low coverage since they are heavily dependent on pathway knowledge which is still incomplete leading to a high rate of false negatives7 Pathway topologybased methods use the same steps as FCS with additional pathway topology information However the reliance on gene overlap leads to similar limitations as ORA and FCSWe could consider the network crosstalk enrichment tools as the fourth generation They rely on a network such as a functional association network like Funcoup8 or STRING9 These networks integrate different experiments from different data types into a single network providing information about gene to gene functional associations which is translated into links in the network With this limitations such as gene independency and low coverage of overlapbased methods are overcome Association between two sets is measured in terms of links between them in the network known as crosstalk In the past few years different ways to assess enrichment between two gene sets have been published like NEA10 EnrichNet11 CrosstalkZ12 NEAT13 NEArender14 BinoX7 and GeneSetDPGeneSetMC15 EnrichNet defines a network enrichment score based on network distances between two gene sets using random walks with restart but is not able to calculate statistical significance Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Box Solna Sweden email eriksonnhammerdbbsuseScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cof the enrichment The tools NEA and CrosstalkZ assess significance using statistical tests assuming that crosstalk between nonenriched gene sets is normally distributed but this is often not the case Moreover they rely on network randomizations to obtain null model parameters which makes them computationally very slow Computational time is reduced in BinoX which also applies network randomization but uses the binomial distribution to calculate statistical significanceThe methods NEAT NEArender and GeneSetDPGeneSetMC do not use network randomization NEAT calculates the expected number of links between two gene sets based on their degrees and then uses the hypergeometric distribution to assess statistical significance NEArender computes the expected number of links in the same way as NEAT but uses a chisquare test to assess statistical significance GeneSetDP uses dynamic programming to calculate an exact distribution of the expected number of links to a pathway for a certain gene set size GeneSetMC does this approximately using MonteCarlo sampling which is faster These two algorithms are however not implemented to allow large scale pathway enrichment analysisThe null model assumption of NEAT NEArender and BinoX is that compared gene sets are expected to behave like random gene sets For real pathways that are very nonrandom eg highly intraconnected this can lead to underestimating the expected level of crosstalk and produce a high false positive rate FPR To avoid this it is important that the method can cope with the nonrandomness of pathways To this end we have developed a novel networkbased pathway enrichment analysis algorithm called ANUBIX Adaptive NUll distriButIon of Xtalk which is based on scoring random gene sets against real pathways to build its null model We show that ANUBIX clearly outperforms recent network crosstalk methods like BinoX NEArender and NEAT in terms of avoiding False Positives FP showing that it can model expected network crosstalk to pathways more preciselyMaterial and methodsOur networkbased pathway enrichment analysis tool ANUBIX depends on a global functional association network We used the network Funcoup version with a link confidence cutoff of containing genes and links With those genes cid31g1 g2 gn gncid30 S and all the pairwise links between them form a symmetric matrix A with dimensions SxS such thataij 1if gi is connected to gj and i � j otherwise aij A gene set Q and a pathway P are a subset of the total number of genes for a certain proteome such that Q P S Notice that S Q we can have some genes from the proteome that are not in the network The crosstalk between Q and P is measured with the degree k cid31iQcid31jPaijThe null model is built based on the expected crosstalk between a random gene set of the same size as the original gene set Q and pathway P Since the network connections are binary each link is considered as a Bernoulli trial Y ¼ Bcid31pcid30 where p is the probability of observing a link We also calculate n QP Q P all the possible links between Q and P We count the links each gene from Q has to the pathway P meaning that if two linked genes are in Q and also in the P we count that link twice boosting the cases where we find overlap Each of these Bernoulli trials are assumed to be independent and the sum of them follows a binomial distributionIn the binomial distribution the mean and variance are defined as µ np and Var npcid311 pcid30 respectively This means that µ ¥ Var which may not be true when the random variable is overdispersed leading to an underestimation of its variance16The betabinomial distribution has been extensively used as an alternative to handle overdispersed binomiallike random variables1718 Here the probability of success p is not fixed as it is in the binomial distribution but follows a beta distribution Betaα β with parameters α and β The marginal distribution of the betabinomial is described in Eq a0fcid31kn α βcid30 cid29 nk cid28 Bk α n k βBα βTo estimate the optimal parameters of the betabinomial we use maximum likelihood estimation MLE19 where the loglikelihood is Eq a0lcid31kn α βcid30 logLcid31kn α βcid30 logcid29 n logcid29 nk cid28 logBα k β n k logBα βk cid28 logŴα k logŴβ n klogŴα β n logŴα logŴβ logŴα βThe negative loglikelihood is optimized with the Nelder and Mead method20 The factorial term in the loglikelihood is removed since it does not depend on the parameters to be optimized Once we have the betabinomial parameters α β of our null distribution we calculate if the crosstalk between Q and P is enriched The null and alternative hypotheses areH0 No more links between Q and P than expected by chanceH1 More links between Q and P than expected by chanceBecause of the discrete nature of the null distributions ordinary pvalues are conservative and therefore mid pvalues were used2122 Mid pvalue is defined as half the probability of the observed statistic plus the probability of observing more extreme values22 The workflow of the ANUBIX algorithm is depicted in Fig a0Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Workflow of ANUBIX The algorithm assesses the significance of the network crosstalk between a query gene set and a pathway A null distribution is generated for each pathway to model the expected crosstalk of random gene sets of the same size as the original gene set This distribution is then fit to a betabinomial distribution to calculate the probability of reaching at least the number of observed links or more between the query gene set and the pathway Software Inkscape version inksc apeIt is important to point out that the networkbased approaches ANUBIX NEAT NEArender and BinoX test three different types of null hypothesis ANUBIX which takes only enrichment into account computes a one tailed test NEAT computes two onetailed tests for enrichment and depletion and takes the minimum pvalue of them multiplied by to emulate a twotailed test BinoX and NEArender compute both enrichment and depletion but only perform one onetailed test since the hypothesis test changes depending on whether the observed number of links is above or below the expected crosstalkPathways To generate the false positive and true positive benchmarks we used KEGG v70123 pathways and REACTOME v6224 pathways for Homo sapiens REACTOME pathways have a deep hierarchical structure including many small pathways on the lower levels that are very specific To reduce Reactomes specificity we resolved its hierarchy by collapsing lower level pathways below a certain pathway size to their parents until obtaining an average pathway size similar to KEGG pathways genes per pathwayPerformance measures In the FP benchmark we generated random gene sets and tested them against KEGG and REACTOME pathways To make these gene sets representative of real experiments we took the average size of MSigDB25 gene sets which is genesIn the True Positive TP benchmark we bisected the KEGG pathways and REACTOME pathways into two parts Each part gets a similar number of genes and links7 To be able to benchmark GEA we emulated some overlap between the two bisected parts This overlap corresponded to the average overlap between the MSigDB gene sets and the pathway measured individually for each of the pathways in KEGG and REACTOMECorrection for multiple hypothesis testing was done using the BenjaminiHochberg procedure26Stability Our null distributions are based on random sampling We take random samples of genes from the genome This stochastic procedure makes the null distributions different every time they are generated Since the pvalues are computed from the null distribution their values may change To analyze stability we generated the null distribution times for the crosstalk between the same gene set to the same pathways for increasing numbers of random samples For each sample size we computed the coefficient of variation CV which is the ratio between the standard deviation SD and the mean We required a CV lower than to limit the dispersion of the mean of the null distribution and this was reached at random samples Once the number of random samples were chosen we measured how much the pvalues were varying in each run For that we ran a randomly selected MSigDB gene set times To compute the confidence interval of the pvalues we used the central limit theorem and applied normal distribution statistics to compute themUsed programs ANUBIX bitbu cketsonnh ammer group anubi xBinoX bitbu cketsonnh ammer group binox NEAT cranrproje ctwebpacka gesneatneatpdfNEArender cranrproje ctwebpacka gesNEAre nderNEAre nderpdfGeneSetDP githu bcomstati stica lbiot echno logygenes etdpScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Overdispersion of KEGG and REACTOME pathways null distributions when sampling random gene sets of size from the proteome The dispersion for each pathway is calculated as the ratio between the variance and the mean of the crosstalk null distribution For each pathway database we illustrate the dispersion values through a boxplot and also by showing the dispersion distribution Software R version wwwrproje ctFigure a0 Observed crosstalk distribution fit with binomial and betabinomial distributions random gene sets of size were used to generate a null distribution of crosstalk to the A Betaalanine metabolism B Prostate cancer and C Alzheimers disease pathways Betabinomial shows a much better fit to the observed link distribution than the binomial Software R version wwwrproje ctResultsTo correctly assess the statistical significance of an observed network crosstalk between two gene sets eg one experimental gene set and one known pathway it is paramount that the null distribution appropriately models the crosstalk of random query gene sets Note that it is not necessarily appropriate to assume that the pathway gene set behaves like a random gene set ie the null distributions need to model crosstalk between random query gene sets versus real pathway gene sets It is also paramount to model the expected crosstalk distribution with an appropriate distribution Previous methods such as BinoX or NEAT use binomial and hypergeometric distributions respectively which are not appropriate for overdispersed distributions since they do not allow the variance of the distribution to be greater than the mean To showcase this we generated null distributions for KEGG and REACTOME pathways by sampling gene sets of size from the proteome In Fig a0 we show the dispersion for each pathway as the ratio between the variance and the mean of the crosstalk null distribution We observe that almost all of these distributions suffer from overdispersion meaning that the variance of the distribution is greater than the mean Therefore statistical models that cannot cope with overdispersion are not appropriate to model the null distribution of most pathwaysTo visualize the overdispersion in detail we chose pathways that are in different quartiles of the dispersion distribution We show their null distributions in Fig a0 Figure a03A shows the Betaalanine metabolism Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Pvalue uniformity test of ANUBIX Binox GEA GeneSetDP NEArender and NEAT random gene sets of genes were tested for crosstalk enrichment against the KEGG pathway Prostate cancer A Reported pvalues are plotted against theoretical quantile rank A perfect method should adhere to the diagonal B Distributions of the FPR for all KEGG and REACTOME pathways tested with ANUBIX BinoX NEArender NEAT and GEA Green distribution for enriched tests and red distribution for depleted The dashed line at FPR denotes the expected FPR level The black triangle and circle represent the mean FPR for enrichment or depletion respectively Software R version wwwrproje ctpathway whose dispersion value is in the first quartile Figure a03B shows the Prostate cancer pathway with a dispersion in the second quartile and Fig a03C shows the Alzheimers disease pathway with a dispersion in the fourth quartile The high variance relative to the mean gives a very poor fit with the binomial distribution yet the betabinomial distribution gives a very good fit This underestimation of variance by the binomial distribution would lead to many false positives With a few pathways there is no overdispersion in the data but these can fit a betabinomial equally well as a binomialBenchmark for false positives Since the null model in ANUBIX is based on random gene sets we expect the pvalue distributions when tested with random query gene sets to behave uniformly for any pathway For almost all pathways we observed a virtually perfectly uniform distribution when plotting ANUBIX pvalues of random gene sets against each KEGG pathway full results at Supplementary Fig a0 A few pathways deviated somewhat from uniform which is the result of the betabinomial fit not being able to model the null distribution with enough precision A second type of deviation from perfect uniform distribution is caused by staggering of observed pvalues This is relatively frequent and arises because the support of the test statistics is limited to a few values and therefore unavoidable We also generated the pvalue distributions for gene sets of size and size against each KEGG pathway Supplementary Fig a0 and respectively which gave similar results However some pathways seem to be affected by the size of the gene set ANUBIX was compared to the top networkbased methods BinoX NEAT and NEArender and a recently published method GeneSetDP For comparison we also tested a popular overlapbased pathway enrichment method GEA Because GeneSetDP and GenesetMC are too computationally heavy for large scale analysis we first tested all the gene sets against one individual pathway We only used GeneSetDP because GeneSetMC produces similar pvalues Pvalues were plotted versus quantiles of a uniform distribution For an unbiased method the pvalues would lie on the diagonal y x Figure a04A shows that for the Prostate cancer pathway Pvalues of ANUBIX adhere to the diagonal much better than for BinoX NEAT NEArender and GEA while performing equally well as GenesetDPFor crosstalk to random gene sets we expect of the pvalues to be lower than However for the Prostate cancer pathway BinoX had of its pvalues lower than NEAT and NEArender GEA whose coverage is small7 had of its pvalues below and highly discrete taking on only four possible values for Prostate cancer due to few overlapping genes ANUBIX and GeneSetDP find a correct fraction of the pvalues with and GeneSetDP under respectivelyWe also ran ANUBIX BinoX NEAT NEArender and GEA for the random gene sets against all pathways in the KEGG database and REACTOME database Full results in Supplementary Data and Data respectively GeneSetDP was not included as it is not implemented to run at a large scale NEAT NEArender and BinoX can also give statistical significance when gene sets have fewer links to a pathway than expected by chance known as depletion To make a more consistent benchmark where all methods can be compared equally we only considered enrichment and depleted pathways were treated as nonsignificant The average FPR for all KEGG pathways was with ANUBIX with BinoX with NEAT with NEArender and with GEA For REACTOME almost the same FPR values were obtained ANUBIX BinoX NEAT NEArender and GEA Roughly the same FPR levels came from significant depletions for BinoX NEAT and NEArender However the averaging of the FPR levels for all pathways does not show the real problem of these methods Some pathways could give very nonconservative pvalues while other pathways could give very conservative pvalues To show how each method performs for each of the pathways we plot the distribution of the FPR fraction of pvalues below for each pathway as violin plots in Fig a04B Since GEA Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cand ANUBIX cannot test for depletion they only have the enriched case A perfect method would have all points close to the dashed line at FPR ANUBIX produces FPR values close to this line meaning that the model is robust GEA greatly underestimates FDR and produces almost no false positives but this leads to very poor sensitivity as shown below NEArender NEAT and BinoX produce similar FPR distributions that are very spread out ie the FPR tends to be very different for different pathways For the tests performed per pathway some pathways reach an FPR of for enriched cases and similar for depleted Summing these two can lead to a total FPR above if we take both enriched and depleted cases into account which is very nonconservative The plot also shows that for some pathways these methods are overly conservative giving considerably lower FPR than they should In other words methods like BinoX NEAT and NEArender have a huge variation in the quality of their pvalues depending on the pathway under studyBinoX is implemented in a web server called PathwAX27 where users can submit a query gene set to test for network crosstalk enrichment By analogy we studied false positive rates assuming independence between gene sets where each user submits a single gene set ie multiple testing correction is only performed for number of pathways each query is compared to random gene sets were used against the KEGG database A FDR threshold of was used and enrichment and depletion were grouped separately as shown in Fig a05A The top pathways with highest FPR for BinoX were plotted full results in Supplementary Data all having a highly nonconservative behaviour for BinoX NEAT and NEArender Every time a user submits a random gene set the chance of getting one of these pathways is very high on average if we take both enriched and depleted cases into account In contrast ANUBIX and GEA have less than FPR We observed a very high correlation between perpathway FPR values for BinoX NEAT and NEArender above for each pairwise comparison This indicates that the pathway enrichment analysis results obtained with these methods are highly similar They all had low Pearson correlation to ANUBIX with for BinoX for NEAT and for NEArender The corresponding Spearman correlations were and As for the pathways we noticed that there is a high overlap between some of them For instance the Alzheimers disease and Parkinsons disease pathways share of their genes The Alzheimers disease the Parkinsons disease and the Huntingtons disease pathways have of the genes in common from the union between them Further the Oxidative phosphorylation the Nonalcoholic fatty liver disease the Alzheimers disease the Parkinsons disease and the Huntingtons disease have of the genes in common from the union between them Therefore if there is significant crosstalk to one of them crosstalk to the other pathways is very likely The high dependency between some pathways points to opportunities for further improvement of pathway definitions Further exploration was performed in these pathways topologies to understand their tendency to generate many FPsWe computed the fraction of intralinks for each pathway as the ratio between the number of internal links and the total number of links We plotted this ratio against the FPR Fig a05B A higher fraction of intralinks means that more links are within the pathway than to the outside suggesting a more isolated pathway The Spearman correlation coefficient between the fraction of intralinks and FPR for BinoX was indicating that the fraction of internal links plays a major role in causing false positives This dependence is also observed with NEAT with a correlation of and with NEArender at However ANUBIX had a correlation of only and GEA This indicates that methods like NEAT NEArender and BinoX cannot deal properly with pathways that are clearly not random and behave more as isolated communitiesAdditionally we calculated the number of maximal cliques each of the KEGG pathways has and we observed a correlation with the FPR for BinoX with a spearman correlation of These maximal cliques were computed using the igraph package in R We considered cliques as all complete subgraphs and a clique is considered maximal if we cannot add more nodes to it This indicates that the higher the number of maximal cliques in a pathway meaning a less random pathway in terms of topology the higher the FPR isBenchmark of true positives Besides a correct FPR it is also important to verify that the power of the method is sufficient for a high true positive rate TPR To this end we devised a benchmark by splitting each KEGG and REACTOME pathway into two parts and then measured each methods ability to reconnect these parts The splitting into parts included giving an amount of gene overlap between the two parts emulated based on the average overlap between MSigDB gene sets and KEGG and REACTOME pathways We compared the methods by their Receiver Operating Characteristic ROC curves Figure a06A shows only the tests that are statistically significant FDR and only considering enrichment ANUBIX has a TPR of of the enrichment tests as significant without having any FP BinoX has a TPR of with FPR NEArender a TPR of with FPR and NEAT a TPR of with FPR GEA whose coverage is low gives only TPR and no FPs Figure a06B shows the ROC curve for all the enriched tests performed also including insignificant results This shows the coverage of each method ANUBIX recovers of the TP tests without suffering any FPs BinoX NEArender and NEAT have similar curves recovering and of the enriched TP tests respectively GEA can here maximally find of the TP tests since only those tests have some gene overlap This benchmark shows that GEA has very low coverage of what it can potentially find We note that the maximal TPR obtained by GEA corresponds to the amount of significantly enriched crosstalks obtained when running all of MSigDB against KEGG pathways see Pathway annotation of MSigDB gene setsStability and robustness Considering that the null distributions are based on random sampling we studied the number of iterations required to reach a coefficient of variation CV of Figure a07A shows how many pathways pass that threshold depending on different amounts of random samples of the pathways had a CV lower than when using random samples to model the null distribution To verify that this number of random samples is sufficient for every pathway we computed the enrichment of one randomly selected MSigDB Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Analysis of why certain pathways are very prone to produce false positives random gene sets of genes were tested independently for crosstalk enrichment against the KEGG pathways A The top ten pathways that produce the highest false positive rate FPR with BinoX and the FPR obtained with other methods B Fraction of intralinks for each of the KEGG pathways against FPR The size of the point reflects the total number of links in each pathway Software R version wwwrproje ctgene set to all KEGG pathways times The null distributions are thus generated times for each pathway and we would expect some changes in the pvalues between runs Figure a07B shows the standard deviation of the pvalues We observe that the pvalues almost did not vary showing that random samples are enough Moreover because of sampling the pvalue is not an exact pvalue but a point estimate of it we also provide with the confidence interval of each of the pvalues Supplementary Data Compute time Our method relies on random sampling to model the null distribution which makes ANUBIX computationally intensive To benchmark its speed we did runs each time with a randomly chosen biological gene set extracted from MSigDB against KEGG REACTOME and KEGG plus REACTOME We measured the compute time for each of the networkbased methods see Fig a0 With this benchmark we can show that ANUBIX is fast when running single gene sets One should take into account that ANUBIX and BinoX need a precomputation step before running the actual analysis However the ANUBIX precomputation step takes around a0s whereas in BinoX it takes around a0min To compute the randomized network for BinoX Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Receiver Operating Characteristic ROC curve For the TP tests each KEGG and REACTOME pathway is divided into two and a TP is interpreted as the crosstalk between two parts from the same pathway For the FP tests random gene sets of size are tested for enrichment against KEGG and REACTOME pathways A ROC curve for only the significantly enriched tests FDR B ROC curve for all enriched tests Software R version wwwrproje ctFigure a0 Stability analysis of ANUBIX A Fraction of KEGG pathways with Coefficient of variation CV below for different number of iterations B ANUBIX pvalues are stabletheir variance is low and proportional to the magnitude of the pvalue A randomly chosen MSigDB gene set DAIRKEE_CANCER_PRONE_RESPONSE_BPA was run times against KEGG pathways Standard deviation of the logpvalue is plotted against the meanlogpvalues for each pathway Software R version wwwrproje ctwe used iterations A drawback for ANUBIX compared to methods like BinoX or NEAT is that the computation time for large scale analyses take more time For instance the time required to compute the large scale pathway annotation study for the MSigDB gene sets against KEGG pathways took a0min for ANUBIX using cores a0min for NEArender a0min for BinoX and a0min for NEAT Compute times were measured on an i77700 CPU a0GHz with a0Gb RAMPathway annotation of MSigDB gene sets We carried out a largescale pathway analysis study by running MSigDB gene sets against KEGG pathways using ANUBIX BinoX NEAT NEArender and GEA Full results are in Supplementary Data In total crosstalk tests were done per method and to get a more fair comparison between different methods we only considered enriched crosstalk considering that ANUBIX and GEA can only test for enrichmentNEArender BinoX and NEAT found the highest number of significantly FDR enriched crosstalks with and of all pairs respectively followed by ANUBIX with and GEA with Many MSigDB gene sets thus appear to have a high occurrence of pathway enrichments Even if we do not know whether those enrichments are TPs or FPs we show above Figs a0 and 5A that BinoX NEArender and NEAT are prone to produce FPsThe Venn diagram in Fig a0 shows that the overlap between BinoX NEAT and NEArender is very high having of their significant pathway annotations in common This was expected since all these methods consider pathways as random The overlap is even higher between NEAT and NEArender because they compute Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Compute time when running a random experimental gene set from MSigDB different gene sets were tested against KEGG REACTOME and KEGG plus REACTOME pathways for each of the networkbased methods Since ANUBIX allows parallelization we also added another run with cores The error bars show the variability in compute time for each of the methods in each of the databases The BinoX precomputation step is not included since it takes a0min Software R version wwwrproje ctFigure a0 KEGG pathway annotation for MSigDB gene sets with five methods The Venn diagram shows the number of shared pathway annotations at FDR Note that ANU | 2 |
EndocrinesyPostoperative vocal fold dysfunction in covid19 era are we still intime for a recoveryElena Bonati Elena Giovanna Bignami2 Paolo Del Rio1Received May Accepted July Springer ScienceBusiness Media LLC part of Springer Nature To the EditorThe novel coronavirus COVID19 is a highly contagious zoonosis produced by SARSCoV2 which arose inChina and spread all over the world transmitting from manto man through respiratory secretions In March itwas deï¬ned by the World Health anization WHO as apandemic to underline its spread and severityHealthcare professionals are one of the categories most atrisk of contracting the infection in particular when theiractivity involves the direct management of the patientsairways Among these categories we can count anesthetistshead and neck surgeons otolaryngologists maxillofacialsurgeons ophthalmologists and dentists For these reasonsthe latest evidencebased recommendations for otolaryngology and head and neck surgery practice suggest thathealthcare facilities should prioritize urgent and emergencyvisits and procedures until this condition stabilizes ceasingelective care []Nevertheless oncological surgical activity althoughslowed down did not stop in most hub hospitals Regardingthyroid cancer thyroid surgery is complex and the rate ofnerve damage is still considerable Immediate postoperativevocal fold rate is in our case study and decrease to after months Postoperative dysphonia can becaused by several factors other than nerve damage such astracheal intubation or scarring in the thyroid lodge It istherefore important to identify the cause of vocal corddysfunction and treat it correctly at the right time If anunilateral vocal fold paresisparalysis is diagnosedthetreatment consist in improving the speech while in case of Elena Bonatiebonati86gmailcom General Surgery Unit Department of Medicine and SurgeryParma University Hospital Parma Italy Unit of Anesthesiology Department of Medicine and SurgeryParma University Hospital Parma Italybilateral vocal fold paresisparalysis respiratory obstructionalso needs to be urgently treated Fortunately we havebroughtthe incidence of this last and most dangerouscomplication to at our Clinic since the introduction in of the routine use of intraoperative neuromonitoringduring thyroidectomyThe latest guidelines published by the Americanin March Association of Endocrine Surgeonsrecommend laryngeal examination in patients with knownor suspected new recurrent laryngeal nerve dysfunctionafter thyroidectomy for additional evaluation and possibletreatment with a speech pathologist According to theAmerican Academy of OtolaryngologyHead and NeckSurgery they assert that early referral weeks postsurgery to a laryngologistin combination with earlyintervention results in superior voice outcomes since theideal time for vocal fold augmentation is months afterthyroidectomy []A metaanalysis about therapy for vocal fold paresisparalysis after thyroidectomy concluded that the timingof therapy for unilateral vocal fold paralysis after thyroidectomy has a significant impact on the effect sizebeing significantly greater if therapy is performed within months This may be explained by progressive atrofolds and disappearance of nervephy offunction so that vocalfold movements cannot berecovered []the vocalPatients who underwent thyroid surgery from February and who had experienced a vocal fold disfunctionVFD were unable to undergo a laryngoscopy nor muchless a speech therapy according to health measuresnecessary to contain the spread of the virus This unfortunately causes a progressively reduced possibility ofrecovery increasing the speciï¬c morbidity related to surgery for thyroid cancer in this period The only indicationthat we can give to patients is the rest of the voice to avoidthe establishment of compensation mechanisms worseningthe clinical picture waiting to be able to resume the correcttreatment 0cTherapeutic diagnostic pathways in the COVID19 erahave become difï¬cult and dangerous logarithms that mustconsider the need for patient care and the possibility oftreatments delay in safety but also the risk of contagion ofthe patientsleast protection ofhealthcare personnel The hospital setup has been significantly changed and much of the economic structuraland human health resources have been dedicated to themanagement of the COVID pandemicthemselves and notIn parallel with the COVID19 emergency we areexperiencing another health emergencythe one thatinvolves the management of nonCOVID19 patients Evenin the second phase of the pandemic only urgent healthservices are provided A reanizing effort within theindividual healthcare companies is required to guaranteetreatment even for nonCOVID19 patientsMoreoverThe COVID19 pandemic highlighted the limits andweaknesses of our health system and now that the correctprotocols for the protection of healthcare personnel havebeen described allthe all healthcare companies shouldequip their staff with the appropriate materials such as N95masks hair cover protective coverall gown gloves faceshields goggles and shoe covers In the face of higherexpenses this would allow the resumption of activitiesminimizing the risk of an increase in the rate of infectionroutine health practices must be reconsidered preferring less invasive techniques in order toscreen patients who need secondlevel examination Evenif not used yet in our hospital transcutaneous laryngealultrasonography is a valid noninvasive and painlessalternative method in the assessment of vocal cords It hasbeen demonstrated in a recent prospective multicentricstudy that it has concordance with laryngoscopy in themajority of cases and so it can be a valid alternative asï¬rstline exam for vocalfold examination pre andpostoperativelyEndocrineFinally the growing use of virtual platforms for the needof social distancing could encourage their application evenin healthcare services that can be performed by teleconference such as speech therapyWe can assess that COVID19 pandemic is causingdirect morbidity and mortality and even a related one dueto missed or delayed treatment of multiple nonCOVID19diseases The delivery of the health service should beimproved and the health system itself must be modernizedto adapt to new needsCompliance with ethical standardsConï¬ict of interest The authors declare that they have no conï¬ict ofinterestPublishers note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional afï¬liationsReferences LP Kowalski A Sanabria JA Ridge WT Ng R de BreeA Rinaldo RP Takes A A Mkitie AL Carvalho CR Bradford V Paleri DM Hartl V Vander Poorten IJ Nixon C PiazzaPD Lacy JP Rodrigo O GuntinasLichius WM MendenhallA DCruz AWM Lee A Ferlito COVID19 pandemic effectsand evidencebased recommendations for otolaryngology and headand neck surgery practice Head Neck 101002hed26164 KN Patel L Yip CC Lubitz EG Grubbs BS Miller W ShenP Angelos H Chen GM Doherty TJ Fahey 3rd E KebebewVA Livolsi ND Perrier JA Sipos JA Sosa D StewardRP Tufano CR McHenry SE Carty The American Associationof Endocrine Surgeons Guidelines for the deï¬nitive surgical management of thyroid disease in adults Ann Surg e21e93 X Chen P Wan Y Yu M Li Y Xu P Huang Z Huang Typesand timing of therapy for vocal fold paresisparalysis after thyroidectomy a systematic review and metaanalysis J Voice 0c' | 2 |
"Cellular recognition of microbial DNA is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens Cyclic GMPAMP synthase cGAS and its downstream effector stimulator of interferongenes STING are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype I interferons IFNs and other inflammatory cytokines Accumulating evidence suggests that the activation ofthe cGASSTING axis is critical for antitumor immunity The downstream cytokines regulated by cGASSTINGespecially type I IFNs serve as bridges connecting innate immunity with adaptive immunity Accordingly a growingnumber of studies have focused on the synthesis and screening of STING pathway agonists However chronicSTING activation may lead to a protumor phenotype in certain malignancies Hence the cGASSTING signalingpathway must be orchestrated properly when STING agonists are used alone or in combination In this review wediscuss the dichotomous roles of the cGASSTING pathway in tumor development and the latest advances in theuse of STING agonistsKeywords cGASSTING Innate immunity Type I interferon STING agonists Antitumor response CancerdevelopmentIntroductionThe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses[]Protection againston patternrecognition receptors PRRs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens Correspondence zqliucsueducn Juyan Zheng and Junluan Mo contributed equally to this work1Department of Clinical Pharmacology Hunan Key Laboratory ofPharmacogenetics and National Clinical Research Center for GeriatricDisorders Xiangya Hospital Central South University Changsha Peoples Republic of China2Institute of Clinical Pharmacology Engineering Research Center for appliedTechnology of Pharmacogenomics of Ministry of Education Central SouthUniversity Changsha Peoples Republic of ChinaFull list of author information is available at the end of the adaptive immunity [] Abnormal RNA or DNA RNADNA hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns PAMPs [ ] Cells associated with innate immunity recognize different microbial PAMPsthrough specific PRRs thereby playing key roles in hostresistance to microbial infection [] The pathways governing RNA recognition such as retinoid acid induciblegene I RIGIlike receptors have been reviewed elsewhere and will not be covered herein In the case of DNArecognition one of the best known PRRs is Tolllike receptor TLR9 which senses extracellular CpG hypomethylated DNA that has entered the cytosol through thephagosomelysosome system [] In addition the AIM2like receptor AIM2 inflammasome can be triggered afterthe entry of doublestranded DNA dsDNA into the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZheng Molecular Cancer Page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as IL1and IL18 and the activation of gasdermin D leading topyroptosis [] Nevertheless the most notable PRR iscGAS a direct cytosolic dsDNA sensor which was identified by Dr Chens group in [] Once cGAS bindsto dsDNA the cGASSTING pathway is activated to further induce the expression of type I IFNs and other inflammatory cytokinesthus triggering innate immuneresponses [] Mounting evidence suggests that cGASSTING signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis Hence in this review we summarize themechanism of cGASSTING activation and elaboratefindings regarding its dual effects on tumor developmentCurrent advances in the use of STING agonists as a novelstrategy for antitumor therapy are also reviewedInsights into the cGASSTING signal transductioncascadecGAS is an innate immune sensor that identifies variouscytosolic dsDNAincluding DNA with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderivedDNA and selfDNA Table In the cytoplasm cGAS isactivated by interacting with dsDNA in a sequence[]independent butStructural and biochemical analyses have revealed thatthe Cterminal lobe of cGAS contains a conserved zinclengthdependent mannerionbinding module that mediates DNA binding andcGAS dimerization [ ] DNA ligands promotecGAS activation primarily by inducing conformationalchanges around the catalytic site and in the DNAbinding structures of cGASthe GScontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cGAS activation byDNA [] In addition to the primary DNAbinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops [] The proximity ofthe two DNAbinding sites in cGAS leads to a cGASDNA complex assembly in which two cGAS moleculesembrace two molecules of dsDNA [ ] The cGASdimers are anized in headtohead alignment nextto the DNA [] and thus form stable ladderlike networks between one long curved dsDNA helix or two independent dsDNA strands [ ] In this way eachindividual cGASdsDNA complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsDNA as more likely to activate cGAS [] In additionlong DNA is more efficient than short DNA in drivingthe liquidliquid phase separation of cGAS and the formation ofcriticallydependent on the concentration of cGAS and DNA inthe cytoplasm [] cGAS and dsDNA are spatially concentratedcGASdimerization and activation [] Once cGAS andcGAS liquidlike dropletsin liquiddropletsistofacilitateTable Classification of the cytosolic dsDNA that activates the cGASSTING signaling axisClassificationSelfDNASource of dsDNAMicronucleiPossible mechanismsRupture of the micronuclei membrane leads to exposureof chromatin DNA that is recognized by cGAS whichactivates the cGASSTING pathwayReferences[]MitochondrionNuclear RNAPathogenderived DNADNA virusHSV1 HSV2 KSHV adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus RetrovirusHIV SIV murine leukemia virusRNA virusWest Nile virus dengue virus VSVSARSCOV2BacteriaListeria monocytogenes Mycobacteriumtuberculosis Listeria Shigella FrancisellaChlamydia and NeisseriaMitochondrial stress induces mtDNA leakage into thecytosol thus activating the STING pathway and inducingproduction of cytokinesFacilitated by endogenous retroelements nuclear RNAcan be reversely transcribed into DNA that activatescGASSTING signaling[][]DNA viruses invade host cells and release pathogenderivedDNA to induce STING activation[]DNA intermediates generated from reverse transcription maybe recognized by cGAS to stimulate downstream STINGsignaling[]Infection with RNA viruses might cause cellular damage andcell death which results in the release of cellular DNA andfurther activation of the cGASSTING axis SARSCoV2 bindingto ACE2 can lead to excessive angiotensin II signaling thatactivates the STING pathway in mice[]Bacteria produce CDNs such as cyclic diGMP and cyclicdiAMP which can directly bind to and activate STING[ ]HSV1 herpes simplex virus HSV2 herpes simplex virus KSHV Kaposi sarcomaassociated herpesvirus HIV human immunodeficiency virus SIV simianimmunodeficiency virus VSV vesicular stomatitis virus CDNs cyclic dinucleotides and SARSCOV2 severe acute respiratory syndrome coronavirus 0cZheng Molecular Cancer Page of dsDNA interacts structural switches rearrange the catalytic pocket to enable cGAS to catalyze the synthesis of²²cyclic GMPAMP ²²cGAMP with ATP andGTP as substrates The first step in this process is theformation of a linear dinucleotide ²pppG ²²pAwith ATP serving as the donor and ²OH on GTP serving as the acceptor Then the intermediate product flipsover in the catalytic pocket placing GTP at the donorposition and AMP at the acceptor position to form asecond ²² phosphodiester bond [ ] Notablyalthough dsRNA or singlestrand DNA ssDNA is ableto bind to cGAS neither can rearrange the catalyticpocket which may explain the exclusive activation ofcGAS by dsDNA Ultimately cGAMP acts as a secondmessenger to bind to and activate STING a small endoplasmic reticulum ERlocated protein KD withfour putative transmembrane domains [ ] Normally in a resting state STING is retained in the ER byinteracting with the Ca2 sensor stromalinteractionmolecule STIM1 [] The cytosolic ligandbindingdomain LBD of STING exists as the most functionalunit capable of integrating with ²² cGAMP or CDNscyclic dinucleotides such as cdiAMP cdiGMP or ²²cGAMP from bacteria Upon interaction the obviousclosure of the ligand binding pocket in the LBD is observed which is related to the activation of STING []Next STING transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe ER to the perinuclear area facilitated by cytoplasmiccoat protein complex II COPII and ADPribosylationfactor ARF GTPases [ ] In the Golgi STING ispalmitoylated atCys88 andCys91 a posttranslational modification necessary forSTING activation [] Modified STING recruits thekinase TANKbinding kinase TBK1 in turn the Cterminal domains of STING are phosphorylated byTBK1 and then phosphorylated STING recruits interferon regulatory factor IRF3 which is also phosphorylated by TBK1 and dimerizes ultimately dimerizedIRF3 enters the nucleus and exerts its function in thetranscription of type I IFNs and interferonstimulatedgenes ISGs [] In parallel STING can also bind toand stimulate IκB kinase IKK to mediate the production of nuclear factorκB NFκBdriven inflammatorygenes Upon signal transduction termination STING istransferred to endolysosomes for degradation [] Considering that cGAMP can be transferred through gapjunctions or delivered in viralexosome packages cGASSTING signaling may be activated in the cytoplasmwithout dsDNA [ ] Moreover newly produced typeI IFNs activate heterodimer interferon receptors IFNAR1 and IFNAR2 through paracrine signaling and thusinduce the transcription of ISGs [ ] In summaryonce virusderived DNA and selfDNA are located intwo cysteine residuesthe cytoplasm they can be sensed by cGAS and a cGASdsDNA complex is formed to catalyze the synthesis of ²²cGAMP with ATP and GTP Then ²²cGAMP and bacteriaderived CDNs induce STING activation and mediate the release of downstream type IIFNs TNFα and IL6 which are prerequisites for antimicrobial defense and antitumor effects The wholeprocess shows that the dsDNAcGASSTING axis canlead to the activation of both innate and adaptive immunity Fig The antitumor functions of the cGASSTINGsignaling pathwayRecent evidence has revealed the close association of thecGASSTING pathway with cancer development Thissignaling pathway is generally regarded as a potent regulator of cancer immunity A STINGmediated immunesupportive microenvironment can hamper malignancyoccurrence []stressbyTumor cell cytosolic dsDNA induces STING activationUnder normal circumstances DNA is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity [] However DNA leaks aberrantly in tumorcells [ ] Cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism [] Under these intense states nuclear andmitochondrial DNA is fragile and easily damaged whichleads to eventual DNA leakage in the forms of micronuclei chromatin fragments andor free telomeric DNA[ ] Chromosomal instability CIN is the primary source of cytoplasmic DNA in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance [] Excessive proliferation of cancer cells results in unstable genomes [] usuallychromosomal missegregation during mitosis Due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner [] The vulnerable membraneof micronuclei easily exposes the inner DNA to the cytoplasm and activates the cGASSTING signaling axis [] Exogenous stimuli such as chemotherapy and irradiation can also cause DNA damage In addition to leakednuclear DNA oxidative stressinduced mitochondrialDNA leakage is another crucial initiator of STING pathway activation Several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death Therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial DNA escape [ ] Other sourcessuch as apoptotic cellderived DNA exosomal DNAExoDNA and transposable elements have also beencharacterized 0cZheng Molecular Cancer Page of Fig The cGASSTING DNA sensing signaling pathway Various DNA derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic DNA sensor cGAS cGAS catalyzes the synthesis of ²²cGAMP in the presence of ATP and GTP then ²²cGAMP bindsto the ER adaptor STING which also can be activated by CDNs derived from bacteria Upon activation STING translocates from ER to Golgicompartments where it activates TBK1 and IKK which phosphorylate IRF3 and IκBα respectively Then IRF3 and IκBα dimerize and enter nucleusinitiating the transcription of Type I IFN TNF and IL6 The primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cGASSTING activation intumor cells [ ]Type I IFNs mediators of STING and adaptive antitumoreffectscGASSTING signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner On the one hand DNA damage can provokeacute STING signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype SASP which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] In contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein BCL2associated X BAX and downregulating the BCL2 apoptosis[] On the other hand STINGsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type I IFNs to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination []STING activation in nonmalignant cells causes tumorsuppressive effects as well STING signaling protectsagainst colitisassociated carcinomas CACs induced byazoxymethane AOM and dextran sulfate sodiumDSS which induce DNA damage in intestinal epithelialcells and further trigger STING activation Downstreamcytokines of STING signaling such as IL1 and IL18prevent neoplastic transformation by facilitating woundrepair More importantly STING signaling can also provoke cytotoxic T cell responses to control tumorigenesis[] Necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor ATFlike BATF3drivenlineage of dendritic cells DCs [] BATF3 DCs take intumorassociated antigens and migrate towardsthe 0cZheng Molecular Cancer Page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific CD8 T cellsThen CD8 T cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells In turn damaged cancercells release more antigens that are further captured byDCs the whole process forms a positive feedback loopcalled the cancerimmunity cycle [] Tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and T cell priming and activation withtumor antigenspecific T cell priming and activationrelying on DCs and type I IFN release [] The involvement of type I IFNs in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate PRR pathways as potential immunomodulators Mice lacking TLR9 myeloid differentiationprimary response gene MyD88 cytosolic RNA sensor MAVS or the purinergic receptor P2X7R maintainintact antitumor immunity responses whereas mice deficient in STING or IRF3 present with impaired CD8 Tcell priming and activation [ ] In fact dying tumorcells can release multiple damageassociated molecularpatterns DAMPs to trigger innate immune responsesin DCs among these released stimuli tumor cellderivedDNA is a pivotal inducer In general the phagocytosis ofapoptotic cells causesimmune silence because ofDNasebased degradation [] Nevertheless tumor cellreleased DNA can be preserved in the DC endolysosomal compartment through an unknown mechanism [] cGAS recognizes DNA invading the cytoplasm andinduces the activation of STING cascades excretion oftype I IFNs and expression of ISGs Additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial DNAmight be packaged in exosomes Exosomal DNAExoDNA animates STING signaling once it is absorbedby tumorinfiltrating DCs [] Finallytumor cellderived cGAMP can also be transferred to host DCs bythe folate transporter SLC19A1 and then directly bindsto STING activating it in DCs [] A recent study moredirectly demonstrated that cellautonomous STING promoted the maintenance of stem celllike CD8 T cellsand augmented antitumor T cell responses and mechanistically cGASSTINGmediated type I interferon signaling reinforced the stem celllike CD8 T celldifferentiation program mainly by restraining Akt activity []Immune cellderived type I IFNs have crucial functions in antitumor immunity control On the one handtype I IFNs boost cross presentation by various mechanisms first they stimulate the maturation of DCs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of MHC I molecules on the cellsurface [ ] finally they accelerate DC migrationtowardslymph nodes where they can crossprimetumorspecific CD8 T cells [] On the other handtype I IFNs drive the expression of multiple chemokinessuch as CXCL9 and CXCL10 both of which are necessary for cytotoxic T lymphocyte CTL transfer and infiltration [] Similarly type I IFNs restrain the defaultimmune suppressive action of regulatory T Treg cellsby downregulating phosphodiesterase PDE4 and upregulating cyclic AMP cAMP [] Consequently typeI IFNs serve as bridges linking the cGASSTING pathway with CD8 T cellmediated antitumor immunityThe antitumor mechanisms of the cGASSTING signaling axis are illustrated in Fig Indeed previous studies revealed that STING activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [] Additionally STING expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent STING expression [] Lower STING expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] Consistentlycompared with that in the MCFG10A mammary epithelial cell line lower STING expression was detected inmalignant breast cancer cellincluding MCF7HBL100 and T47D cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] Collectivelythat cGASSTING signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinesSTING pathway agonists as cancer therapeuticsThe immunostimulatory potential of the cGASSTINGpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmentTME can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectorT cells Recent drug research has focused on the development of STING agonists because of their potential inanticancer therapy [ ] To date various kinds ofSTING agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates DMXAA and its analogsand small molecular agonists In addition some conventional antitumor therapeutics can also indirectly activateSTING such as chemotherapy radiotherapy RT andtargeted therapy [] In addition STING agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination STING 0cZheng Molecular Cancer Page of Fig The antitumor immunity effect of the cGASSTING pathway DNA damage leads to the formation of dsDNA in tumor cells upon itsstimulation STING signaling is activated and promotes the release of Type I IFN which is crucial for DC maturation STING signaling activation inDCs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cGAMP gap junctions Then DCs migrate towards the tumordraining lymph node and crossprime tumor specific CD8 T cells withthe help of Type I IFNs Finally T cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowCyclic dinucleotides CDNsCDNs constitute a main type of STING agonist whichmainly originate from bacteria The known naturalCDNs consist of exogenous cyclic diGMP cdiGMPcdiAMP ²²cGAMP and endogenous ²²cGAMPAmong these cdiGMP cdiAMP and ²²cGAMPare synthesized by bacteria and identified as secondarymessengers that mediate STING signal transduction inprokaryotic cells while ²²cGAMP functions as theinitiator of STING in mammalian cells [] The antitumor potential of these natural dinucleotides was firstproven by the finding that cdiGMP could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomaH508 cells was inhibited with μM cdiGMP []Intraperitoneal ip injection of highdose cdiGMPdirectly activated caspase3 and triggered T1 tumoripcell apoptosis in vitro nmol of cdiGMP reduced thegrowth of T1 tumor cells in vitro by and nmreduced it by while lowdose cdiGMP nmol accelerated the adaptive T cell response by converting a subgroup of myeloidderived suppressor cellsMDSCs into immune stimulatory cells producing IL12injection of ²²cGAMP [] Consistentlymgkg expedited dramatic leukemic elimination in ElTCL1 transgenic mice bearing chronic lymphocyticleukemia CLL and promoted tumor shrinkage of multiple myeloma in vivo [] From the perspective of endogenous CDNs ²²cGAMP mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing CT26 colon adenocarcinomain a dosagedependent manner relying on DC activationand T cell crosspriming [] More recently OhkuriT further demonstrated that intratumoral it injection of ²²cGAMP μg25 μLdose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0cZheng Molecular Cancer Page of cancer T1luc squamous cell carcinoma mSCC1colon cancer CT26 and melanoma B16F10 mousemodels [] Notably the it injection of ²²cGAMPinhibited not only tumor growth but also lung metastases in mice bearing B16F10 cellderived tumors suggesting that cGAMPinduced CD8 Tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth []termedvaccineSTINGVAXConsidering the superior properties of STING signaling in activating adaptive immunityit is rational toutilize STING agonists such as CDNs as cancer vaccineadjuvants to increase tumor immunogenicity [] Fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor GMCSF and bacteriaderived or synthetic CDNs Theyobserved that after it injection of STINGVAX with μg of CDNs per vaccine dose the volume of B16melanoma tumors was dramatically reduced in a dosedependent manner Compared to mice receiving GMCSF cancer vaccine alone STINGVAXtreated mice hadmore infiltrating CD8 IFNγ T cells in the tumormicroenvironment The in vivo antitumor effect of STINGVAX was also verified in models of colon carcinomaCT26 pancreatic carcinoma Panc02 and upper aerodigestive squamous cell carcinoma SCCFVII []Although natural CDNs are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting First native CDNs are easily degraded by enzymes inside the cellor in the bloodstream Second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede CDNs from penetrating cell membranes to activate cytosolic STING leading to low bioavailability andpoor retention of the CDNs in specific cells and tissuesThird unintentional toxicities and narrow therapeuticwindows are also unavoidable Thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening [] Regarding agonistdelivery Smith reported that biopolymer implantscodelivering cdiGMP μg and chimeric antigen receptor T CART cells resulted in significant tumor regression in mice bearing pancreatic tumors[]Moreoveriv administration of cdiGMPYSK05Lip equivalent to μg of cdiGMP aYSK05liposome delivery system encapsulating cdiGMP led to a tremendous decrease in metastatic lesionsin a B16F10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced [] Chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cGAMP cGAMPNP could activate the STING axis more effectively than solublecGAMP and converted the immunosuppressive TME toa tumoricidal state in a transplanted B16F10 cell melanoma model and in a genetically engineered triplenegative breast cancer model [] Moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of STING agonistsIntroduction of a dinucleotide cyclasecoding gene intothe Escherichia coli Nissle strain was an attempt at realizing this effect however advancements to the systemare needed []tobysnakeApartdigestionresistancecompoundatoms The modifiedfrom improving delivery methods CDNswith superior properties are currently being synthesized and tested For instance to prevent enzymatichydrolysis of cGAMP the nonbridging oxygen atomsin cGAMP phosphodiester linkages were replaced by²²sulfurcGsAsMP showed resistance against degradation byENPP1 a major ²²cGAMP hydrolasetherebyleading to a longer halflife and sustained high affinity for human STING hSTING[] Syntheticdithio mixedlinkage CDNs with both Rp Rp R Rand Rp Sp R S dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforSTING A novel superior modified product ML RRS2 CDA also termed ADUS100 had the potencyto activate all hSTING variants and mouse STINGmSTING ADUS100 had higher efficiency in activating STING signaling than endogenous or exogenous CDNs mainly because of its enhanced stabilityand lipophilicity Its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding B16 melanoma T1 breast cancer and CT26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of ADUS100 three mg doseswhen tumor volumes reached mm3 [] TheremarkableforhSTING laid the foundation for its clinical use Related clinicaltrials of ADUS100 are outlined inTable In addition to ADUS100 some other novelSTING agonists have been well designed IACS8779and IACS8803 are two highly potent ²²thiophosphate CDN analogs that induced striking systemicantitumorin a B16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with ADUS100or cGAMP [] The characteristics and preclinicalapplications of all these mentioned CNDs are summarized in Table Because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of CDNs have beenand high affinityresponsescurativeeffect 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonistsClassificationCharacteristicsApplicationmodelsNatural CDNagonistscdiGMPPoor membrane permeabilitysuitable for various codeliverytechnologiesColon cancer H508cells T1 metastaticbreast cancerTreatmentinformation μM nmol ip nmol ip nmol ip²²cGAMP²²cGAMPHigher binding affinity formSTING than for hSTINGHigher affinity for hSTING thanits lineage isomers binds tovarious STING nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembraneChronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipCT26 colonadenocarcinoma mgkgbreast cancer T1lucsquamous cellcarcinomasmSCC1 μg25 μLdose it μg25 μLdose itcolon cancer CT26 μg25 μLdose itmelanoma B16F10 μg25 μLdose itTherapeutic effects References[ ][][ ]Inhibitsproliferation tumorregression tumorregressionAccelerates TcellresponseLeukemiaeliminationSuppressesgrowthRestrainstumorigenesisImproves survivalrateDelays tumrowthDelays tumrowthDelays tumrowthDelays tumrowthSTINGVAXSyntheticCDNagonistsPotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercGAMPNPsBiopolymer scaffolds cdiGMP and CAR T cellscdiGMPYSK05Lip²²cGsAsMPADUS100IACS8779IACS8803NonCDNagonistsFAALiposomal nanops NPsdeliver cGAMP intracellularlymore effectively than realizedwith soluble cGAMPEradicates tumors moreeffectively than systemicdeliveryYSK05 is a lipid that can efficientlydeliver cdiGMP to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapeMore resistant to degradation byENPP1 tenfold more potent atinducing IFN secretion potentialuse as a cancer vaccine adjuvantImproves stability and lipophilicityhigher affinity for hSTING thannatural CDN agonists capable toactivate all hSTING variants andmSTINGStimulates a superior systemicantitumor response thanADUS100 and cGAMPCauses hemorrhagic necrosisfailed in a phase I clinical trialdue to species specificity μg CDNs itReduces tumorvolume[]B16 melanomacolon carcinomaCT26pancreaticcarcinoma Panc02B16F10 melanomaivTNBCCreates atumoricidal state[]Pancreatic cancer μg cdiGMPTumor regression[]B16F10 mousemelanoma μg cdiGMP ivDecreasesmetastasisTHP1 monocytesB16 melanomathree mg doses it T1 breast cancerthree mg doses itMC26 colon cancerthree mg doses itDurable tumorregressionDurable tumorregressionDurable tumorregression[][][]B16 melanoma μg on day and posttumor implantationAntitumorresponse[]Murine colontumorsExtensive tumorrejection[ ]DMXAAFirst discovered as a vascularRat mammary mgkg ipHigh anticancer[ 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonists ContinuedClassificationCharacteristicsApplicationmodelsTreatmentinformationInduces proinflammatory cytokinesin a STINGdependent mannerHuman fibroblastsAntiviral activity[]Selectively induces STINGdependentsynthesis and secretion of bioactiveIFNs no evidence of binding directlyto STINGActivates STING | 2 |
"it is well understood that the level of molecular oxygen o2 in tissue is a very important factor impacting both physiology and pathological processes as well as responsiveness to some treatments data on o2 in tissue could be effectively utilized to enhance precision medicine however the nature of the data that can be obtained using existing clinically applicable techniques is often misunderstood and this can confound the effective use of the information attempts to make clinical measurements of o2 in tissues will inevitably provide data that are aggregated over time and space and therefore will not fully represent the inherent heterogeneity of o2 in tissues additionally the nature of existing techniques to measure o2 may result in uneven sampling of the volume of interest and therefore may not provide accurate information on the average o2 in the measured volume by recognizing the potential limitations of the o2 measurements one can focus on the important and useful information that can be obtained from these techniques the most valuable clinical characterizations of oxygen are likely to be derived from a series of measurements that provide data about factors that can change levels of o2 which then can be exploited both diagnostically and therapeutically the clinical utility of such data ultimately needs to be verified by careful studies of outcomes related to the measured changes in levels of o2k e y w o r d sclinical measures of oxygen oxygen in tissues partial pressure1department of radiology dartmouth medical school hanover nh usa2department of medicine section of radiation oncology dartmouthhitchcock medical center lebanon nh usa3thayer school of engineering dartmouth college hanover nh usa4department radiation and cellular oncology university of chicago chicago il usa5department of surgery dartmouthhitchcock medical center lebanon nh usa6louvain drug research institute universit catholique de louvain brussels belgium7department radiation oncology university medical center university of freiburg freiburg germany8german cancer center consortium dktk partner site freiburg german cancer research center dkfz heidelberg germanycorrespondenceann barry flood clinepr llc river road lyme nh usaemail annbarryflooddartmouthedufunding informationmajor funding for this work was from the national institutes of health national cancer institute ppg grant p01ca190193 r01 ca p30 ca023108 and national institute of biomedical imaging and bioengineering p41 this is an open access under the terms of the creative commons attribution license which permits use distribution and reproduction in any medium provided the original work is properly cited the authors physiological reports published by wiley periodicals llc on behalf of the physiological society and the american physiological societyphysiological reports 20208e14541 1014814phy214541 wileyonlinelibrarycom phy2 of 0c of introduction the overall goal of this review is to facilitate clinically effective use of measurements of molecular oxygen o2 in tissues with the explicit intent of improving clinical care that is improving the accuracy and effectiveness of diagnoses treatments and prognoses for individual patients this review focusses especially on improving personalized medicine and outcomes of care by carefully considering the basis and validity of clinical measurements of o2 in tissues and how those measurements can be used to advance diagnosis and therapy while measurements of o2 in tissues have been recognized as an important factor in the clinical evaluation and treatment of many diseases especially cancer busk overgaard horsman a0 and pathologies involving ischemia such as in peripheral vascular disease and wound healing insufficient attention often has been paid to the meaning of the values that have been obtained note this review is derived in part from a series of recent papers on this topic flood et a0al a0 swartz flood swartz vaupel instead all too often when a measurement technique has indicated that the level of o2 in a given tissue is x that is is some specific quantitative number for the o2 in the tissue researchers and clinicians alike assume that x is a reliable accurate representation of the true oxygenation status of the tissue this approach ignores the complexity and dynamics of o2 in living biological systems the reality is that any o2 measurement has been taken at only one point in time of a distribution in the subvolume that was interrogated by the method while the o2 is in fact varying with time and across space in the tissue and is unlikely to be uniform in the volume that is being interrogatedin this review we focus on the biologicalclinical meaningfulness of o2 measurements made in living anisms while recognizing that tissue o2 is in constant flux we emphasize that to obtain maximum clinical utility of the measurements it is necessary to consider the goal of the measurements and the limitations of the data that are obtained we particularly focus on the clinical value of making repeated measurements of o2 especially in association with strategiesevents that potentially change o2 levels what are oxygen levels physical concepts and terminology for in tissues reporting on oxygen levels in tissuesthe level of molecular oxygen that is o2 is usually reported as partial pressure of oxygen po2 or concentration of oxygen [o2] or co2 these terms have physically rigorous meanings that can usefully be extended to describe gases such as o2 that are dissolved in liquids or solids including tissues partial pressure is the pressure exerted by oxygen in a mixture of gases while concentration is the content of oxygen in the gas mixture or solid partial pressure is commonly expressed in mmhg and these units are sometimes referred to as torr or kpa si unit used in the eu while concentration of o2 is commonly expressed in ml of o2 per a0ml for example in bloodhowever the solubility of oxygen varies greatly in different media bennett swartz brown koenig a0 jordan et a0al a0 and this affects the relationship between po2 and [o2] the transport of o2 across lipid membranes is known to depend on both diffusion and solubility in the bilayer and to be affected by changes in the physical state and by the lipid composition especially the content of cholesterol and unsaturated fatty acids for example because o2 partitions preferentially into lipophilic media such as membranes the solubility of o2 in membranes is about four times greater than in aqueous solutions m¶ller et a0 al a0 this difference has significant consequences for physical and chemical interactions involving molecular oxygen in biological systems because these interactions depend on the number of oxygen molecules that are present and their rate of diffusiondoes it matter clinically to know whether the technique is reporting [o2] or po2 while these measures are not identical there is a known relationship between them according to the ideal gas law po2 is directly proportional to concentration assuming the volume and temperature are constant that ispv nrtwhere p a0 a0pressure po2 v a0 a0volume n a0 a0amount of substance [o2] r a0 a0ideal gas constant t a0 a0temperatureit is less straightforward in biological systems if the solubility of oxygen in each component of tissue is known and this is not always fairly readily derived experimentally and po2 can be measured then it is relatively straightforward to calculate [o2] conversely if the component in which [o2] is measured and the solubility of o2 in that compartment is sufficiently known then it should be feasible to determine po2 however it often is not feasible to measure these parameters readilybecause of the biological complexities in assessing o2 in tissues each reported measure of o2 level in a tissue can be better considered as an average value average is used here in its more colloquial usage rather than as a statistically defined term because different techniques output their measurement of o2 using differing methods pertinent to that technique each technique gathers information from a particular volume of tissue irrespective of whether that volume is well characterized which we refer to hereafter as the interrogated swartz 0cvolume the sampling of data within the interrogated volume is then used to produce a measure based on a sort of average o2 within that volume characterizing that average measure is made difficult both by the imprecision of knowing the exact volume queried but also because the detection of o2 in that volume may be affected by factors such as its distance from the detector or for optical techniques different rates of scattering that also may not be well characterized hence we conclude that it is important to bear in mind that the measurements of o2 in vivo are fundamentally based on a sort of average within the interrogated volumebecause of all of these challenges in obtaining precise measurements of relevant parameters necessary to assess whether the data obtained by any technique is truly measuring either po2 or [o2] and because of the imprecisions of knowing the volume being assessed and the tissues within that volume it is more realistic to acknowledge the complexity of these issues for in vivo measurements in tissues by using a less precise term for measures of molecular oxygen in tissues such as o2 levels which is the convention we follow in this review we also argue that while it is important to recognize the biological imprecisions in these measures there are still many clinically viable uses of this information such as assessing change in o2 levelsnote too within this paper while focusing on the uncertainties due to sampling issues of each technique and variations due to biological factors we are not taking into consideration further uncertainties due to inevitable instrumental noise variations in the placement of the detector etc expected levels of o2 in tissues table a0 presents some illustrative data on the o2 levels in various tissues both in normal states and as altered by some diseases these measures are presented here as reported in the literature the first column presents the median po2 obtained using the eppendorf electrode or comparable polarographic techniques to measure o2 levels in patients also presented are two other indications of o2 levels the hypoxic fraction the percentage of measurements in a given type of tissue that is below a defined hypoxic level in this case a0mmhg and the range of po2 values found experimentally these data illustrate both the variation in median o2 levels between types of tissues and how they may vary with physiology or disease for example intertissue in general the median o2 levels are lower in skeletal muscle and heart compared to the spleen intratissue the median o2 in skeletal muscle at rest is higher than with exercise while in contrast there is almost no variation between the normal spleen and with hodgkin's disease the data in column illustrate the wide range that any given measurement can have in the same type of tissue that is almost all tissues range from of to a0mmhg even when their median value is quite different see again spleen vs bone however these very high values in the upper range may include experimental artifacts due to the measurement being taken within or very close to an arteriole for example a vessel feeding the microcirculatory bedtable a0 presents the same types of information for a more detailed analysis of changes in an important pathology cancer where o2 levels have been an especially important focus for informing clinical treatment and prognosis to give the reader a sense of how well supported the numbers are the data in table a0 have been ordered by the number of patients included in each rowof interest here all seven cancer types with at least patients studied have a fairly consistent and fairly hypoxic median o2 level a0mmhg similarly all but soft tissue sarcoma have a similar hypoxic fraction sarcoma appears to be about half that glioma appears to be an outlier on the range glioma had no patient whose o2 level was above while all others as was true for the tissues in table a0 have at least one measurement in the upper 80s or 90s these occasional high readings are not surprising since it is plausible that randomly some readings will have been obtained in or very near to arteriolesfeeding microvessels in contrast to the first seven cancers the types of cancers with fewer than patients appear to be more varied in their o2 levels but this is possibly due to being based on few patientsnevertheless even though as noted elsewhere in this review the data presented are not unconditionally absolute values of o2 levels as they are sometimes referred to eg koch a0 macnab gagnon gagnon minchinton fry a0 nevertheless as argued in this review they can provide very useful data as long as their limitations are recognized by researchers and clinicians heterogeneity of distributions of levels of oxygen in tissues examples and causesheterogeneity of distributions of oxygen values in the tissues of interest exists over many dimensions including time and space and over a wide range of scales harrison vaupel a0 tables a0 and focus on intertissue and intratissue variation in overall levels of o2 figures a0 and illustrate heterogeneity using more refined data points to illustrate the skewed nature of the data particularly for malignancies the data in figure a0 are based on multiple measurements made in a series of patients using a computerized polarographic microsensor technique which enables direct assessment of the o2 levels with an o2sensitive needle electrode subject to the limitations of providing true absolute values as discussed swartz 0c of table oxygenation status of anstissuesantissuekidneycortexouter medullainner medullaliverpancreasspleennormalin hypersplenismhodgkin's diseasemyocardiumsubepicardialsubendocardialmucosaoralrectallarge bowelbreastnormalfibrocystic diseaseprostateuterine cervixsubcutisbonecorticalhematopoietic marrowadipose marrowskeletal musclerestingexercisehypovolemic shockpaodskinthermoneutral conditionscritical limb ischemialimbs venous diseasebraingray matterwhite mattermedian po2 mmhghf po2 range mmhgreferencesnananananananag¼nther aum¼ller kunke vaupel and thews samesamekallinowski and buhr 1995akoong vaupel wendling thom and fischer wendling vaupel fischer and br¼nner samewinbury howe and weiss moss for bothkallinowski and buhr 1995asamesamevaupel schlenger knoop and h¶ckel vaupel and harrison samevaupel and kelleher h¶ckel schlenger knoop and vaupel samespencer et a0al a0samesamelandgraf and ehrly jung kessler pindur sternitzky and franke harrison and vaupel landgraf schultehuermann vallbracht and ehrly carreau et a0al a0harrison and vaupel clyne ramsden chant and webster vaupel samecontinuesswartz 0c of table continuedantissueretinamedian po2 mmhghf napo2 range mmhgreferenceshogeboom van buggenum van der heijde tangelder and reichertthoen linsenmeier and zhang white adipose tissuenonobeseobesenanapasarica et a0al a0 hodson lempesis van meijel manolopoulos and goossens abbreviations arterial hf25 hypoxic fraction ¡ fraction of po2 values ¤ a0mmhg na information not available paod peripheral arterial occlusive diseaseelsewhere in the paper oxygen was measured along several electrode tracks in each individual during a given measurement session from near the tumor surface up to tissue depths of a0 a0 a0mm in breast cancers and in cancers of the uterine cervix each row in figure a0 presents a type of tissue breast and uterine cervix with comparisons of o2 levels made in normal tissue green versus malignancies prior to treatment of these ans red the summary measures median po2 values parallel to data reported in tables a0 and are included in the text boxesnote that the data in figure a0 are not normally distributed the distribution of o2 levels made in normal breast tissue is the closest approximation to a normal distribution comparing the two distributions for malignancies we see that breast cancer is more highly skewed than cervical cancer although they have the same median thus using a single measure such as the median could overlook potentially important clinical informationsimilarly comparing the distributions for the normal tissues the normal cervix had a substantial number of o2 measurements within a hypoxic range defined as ¤ a0 mmhg while there were no hypoxic measurements in the normal breast while figure a0 does not differentiate the measurements made per patient it illustrates why several authors report the hypoxic fraction when trying to capture a meaningful overall number to characterize a tissue finally these data underscore why it is important to understand what is well captured by a given measure of o2 leveland what is missed or obscuredanother example of heterogeneity is presented in figure a0 these o2 measurements were taken in a breast cancer patient using epr oximetry with carlo erba ink as the o2 sensor flood et a0al a0 jeong et a0al a0 the data are presented as line widths because the epr oximetry technique using india ink as a sensor undoubtably gathers data from volumes too large to have homogenous oxygen levels however because this sensor remains in the same place in the tissue it still can provide very useful indications of changes over time andor the impact of interventions such as breathing enriched oxygen the data were taken in 30min sessions during which epr spectra were collected continuously but the period was divided into three 10min periods differing by the gas mixture the patient was breathing room air red baseline o2 delivered by a nonrebreather mask green followed by again breathing room air blue recovery the sessions were repeated approximately weekly throughout the period of radiation therapy thus mimicking a clinical course of radiation therapy although there was no attempt to impact therapy in this studythe data for this particular patient illustrate that the o2 levels responded to the patient's breathing an hyperoxic gas mixture and then returned rapidly to the baseline level after the 10min period in this patient there appeared to be some variation across the weeks of treatment with the final levels for each period baseline o2 and recovery all being slightly higher than at the beginning of radiation therapy based on nonoverlapping standard deviations of the first and last measurements causes of heterogeneity in normal tissuesthere are spatial variances in oxygen levels in normal tissues due to the longitudinal gradient in oxygen as the blood passes through the microcirculatory bed decreasing from the arterial inlet to the outlet of the microvessels erickson after the oxygen leaves the microvascular networks the partial pressure of o2 decreases due to radial gradients that is o2 diffuses through the tissues as it gets further from the vessels due to o2 being consumed by the cells as a result there are variations in o2 levels from cell to cell according to their distance from the microvessel within the cells o2 decreases in a microspatially complex manner as it is intracellularly consumed with most of the consumption occurring in the mitochondria there is growing evidence that diffusion of o2 into the cell may be constrained that is that o2 does not freely and rapidly flow into cells across the membrane and therefore there are gradients from outside to inside of cells khan et a0al a0 kurokawa et a0al a0 pias a0these variations of o2 levels that is gradients between and within cells cannot currently be measured as detailed below in discussing temporal variations even if such measurements of spatial heterogeneity could be made they would still be inadequate to understand the full complexity of heterogeneity of o2 for example in some normal tissues there is additional significant macroscopic heterogeneity of o2 swartz 0c of table pretherapeutic oxygenation status of human tumorstumor type ordered by no of patientscervix cancerhead and neck cancerprostate cancersoft tissue sarcomabreast cancerglioblastomavulvar cancermedian po2 mmhgno of patientsrectal cancerlung cancermalignant melanoma metastaticnonhodgkin's lymphomapancreas cancerbrain metastasesnahf po2 range mmhgliver metastasesrenal cell carcinomagall bladder cancerbile duct cancerabbreviations hf25 hypoxic fraction ¡ fraction of po2 values ¤ a0mmhg na information not availablenanananareferencesvaupel et a0al a0vaupel vaupel data synopsesthese ref apply to allabove the linevaupel thews mayer h¶ckel and h¶ckel vaupel mayer and h¶ckel stone et a0al a0kallinowski and buhr 1995a mattern kallinowski herfarth and volm falk ward and bleehen le et a0al a0lartigau et a0al a0powell et a0al a0koong graffman bjork ederoth and ihse rampling cruickshank lewis fitzsimmons and workman kallinowski and buhr 1995a 1995blawrentschuk et a0al a0graffman et a0al a0graffman et a0al a0over space because of their physiology as a consequence of substantial differences in vascularity blood flow and oxygen consumption eg macroscopic heterogeneity between gray and white matter of the brain between subepicardial and subendocardial layers of the myocardium and between renal cortex and renal inner medullathere also are temporal changes in o2 levels in normal tissues griffith a0 moreover the supply of o2 can vary periodically due to rhythmic changes in microcirculatory blood flow which is reflected at all levels from the inflow arteries to the microcirculation finally within the microcirculation there are variations in microvascular flow due to regional regulation in response to varying metabolic demands kimura et a0al a0 important differences in o2 solubility across tissues affecting the relationship between po2 and [o2] were discussed earlier impact of pathology on heterogeneity of o2 levelsthe presence of pathology often significantly increases the amount and extent of oxygen heterogeneity both spatially and temporally vaupel harrison vaupel mayer a0 the presence of pathology often impacts the structuremorphology of the vessels in tumors there often is a significant amount of neoangiogenesis which results in much less ordered and less functional blood vessels busk et a0al a0 the resulting vessels are much less efficient in delivering blood and also tend to be much more prone to leak leakage from these vessels can cause increases in the interstitial pressure which can reduce the effectiveness of the microcirculation due to reduction of the perfusion pressure within the tumor capillaries fukumura duda munn jain a0pathological changes also can result in altered consumption of o2 in malignant tumors o2 consumption is likely to decrease due to poor oxygen delivery andor because of a switch to glycolysis due to metabolic reprogramming ie the warburg effect as a consequence of hif1α overexpression upregulation of oncogenes downregulation of suppressor genes and activation of certain signaling pathways vaupel multhoff a0 vaupel schmidberger mayer a0 pathology can also impact the integrity of the blood vessels for example tumor growth may physically impinge on the integrity of the blood vessels and the swartz 0c of figure distributions of multiple o2 levels made in patients with normal versus malignant tissue breast and cervix figure adapted from vaupel mayer a02017b p figure repeated o2 level measurementsa during each measurement session and over a0days breast cancer patient measured in skin and superficial breast tissue within the radiation field during a course of radiation therapy figure adapted from flood et a0al a0 p afor carlo erba ink epr line width increases with increasing o2 level but the relationship is nonlinear and can be impacted by several factors therefore the data are given here as line widthmetabolic abnormalities in diabetes can impact the structure of blood vessels causing either microangiopathy andor macroangiopathy the results of these processes can produce very significant local variations in the availability of fully functional vascular structures resulting in locally hypoxic regionsswartz 0c of pathology also can impact temporal changes of oxygen and the response to treatment the presence of pathology especially cancer eg acute and cycling hypoxia in cancers and peripheral vascular disease can result in significantly greater variability in o2 levels braun lanzen dewhirst a0 these include shortterm changes especially associated with the structural abnormalities of the microcirculation resulting in increased local variability in flow and longterm changes that develop over time such as those due to disease progression and responses to therapy baudelet et a0al a0 kimura et a0al a0 konerding fait gaumann a0 matsumoto there also is a potential for pathologies to interrelate with each other for example anemic hypoxia can develop in tumors due to the underlying systemic anemia of the patient vaupel mayer a0in addition to these underlying effects of pathology on tissue oxygen levels any applied therapeutic interventions are very likely to induce changes for example cell killing due to radiation or chemotherapy will alter oxygen consumption patterns these same therapies will also affect the o2 supplying vasculature via both antiangiogenic effects andpossiblynormalization of vessel structure jain a0 the effects of therapies will generally vary both spatially and temporally reiterating the complexity of meaningfully characterizing tissue oxygen levels analysis of the ability of clinically available techniques to directly measure levels of o2 in tissues andor resolve the heterogeneity of o2 distributions in tissuesalthough many techniques are often thought to measure actual o2 in tissues only a few actually have the potential to make o2 measurements directly in the tissues of interest springett swartz a0 tatum techniques that can potentially assess o2 directly in tissues include epr epel et a0al a0 swartz et a0al a0 swartz the eppendorf electrode vaupel h¶ckel mayer a0 some optical methods based on direct measurements of target molecules in tissues for example phosphorescence quenching of optical sensors placed directly in tissues or as part of a physical probe such as the oxylite wen et a0al a0 and nmr relaxation techniques colliez et a0al a0two other types of measurements assess o2 in the vascular system blood gases do this directly while optical methods that measure both hemoglobin saturation and total hemoglobin especially near infrared spectroscopy [nirs] scheeren schober schwarte a0 provide a plausible link to the po2 in the bloodhowever the techniques most often used clinically to characterize tissue oxygenation do not in fact measure o2 directly instead they measure indirect parameters that can be plausibly linked to actual o2 levels but only under appropriatedefined circumstances this latter group of techniques includes positron emission tomography pet imaging of glucose derivatives neveu et a0al a0 pet imaging of drugs that localize in hypoxic tissues tran laser doppler flow measures of metabolites that may be affected by o2 levels for example lactate and redox intermediates and several magnetic resonance imagingnuclear magnetic resonance mrinmr blood oxygenation level dependent bold imaging baudelet gallez a0 and mri egeland et a0al a0 note if their basis is understood and the data considered accordingly these can all provide clinically and physiologically useful information even though they do not provide direct information on the amount of o2 in the tissues direct measures of o2 in targeted tissues that potentially can be used in human subjectsthese are techniques that while they have the capability of providing direct quantitative measurements of o2 in homogeneous media cannot provide such data in tissues in vivo because the volumes that they sense are larger than the volumes of homogeneity of o2 in actively metabolizing tissues consequently all in vivo measurements of o2 are inherently averages of the actual oxygen content in that volume even neglecting the need to include measures of heterogeneity inside cells based on the usual volume of cells and assuming that differences are sought for aggregates of ¤ cells for a measurement of heterogeneity sensed within a a0mm diameter volume the spatial resolution needed to appropriately characterize o2 levels in this volume becomes million voxels the measuring techniques may not even provide a welldefined averaged po2 value within the volume that they sense for example sensors for the signal that is being measuredin the next sections we review the characteristics of each technique that can directly measure o2 levels in tissues we also briefly remark on the volumes they measure and how the measures obtained can be useful clinically see also ortezprado dunn vasconez castillo visco epr oximetry using appropriate particulate paramagnetic materials epr oximetry can provide direct measurements of o2 that is the epr signal is directly proportional to the amount of o2 epel bowman mailer halpern a0 swartz vaupel swartz 0cthe because each multisite sensor senses a volume that is much larger than capillary networks these techniques provide a volume averaged sampling of all compartments within the tissues the time resolution of the techniques can be milliseconds or shorterthe measured parameter of an epr spectrum that indicates the amount of o2 present is the line width of the observed resonance peak there usually is a fixed relationship between the line width and the amount of o2 with the relationship being specific for each type of paramagnetic material for example carbon charcoal or phthalocyanine particulates using particulate oximetric materials measurements can be continuous over any span of time and can be repeated indefinitely see example in figure a0 the method requires that the sensing material be injected or implanted in one or more regions of interest but thereafter all measurements can be made entirely noninvasively importantly the measurements can be carried out in a clinical setting and can fit into the workflow needed for patient careinitial clinical epr measurements of oxygen in tissues have used india ink as the oxygen sensor swartz et a0al a0 the carbon ps are the components that respond to oxygen lan beghein charlier gallez a0 after injection of a0µl of the suspension through a small needle the carbon ps disperse nonuniformly through the local region as small extracellular aggregates they are often engulfed by macrophages the resulting epr spectra in the region probed by the resonator ie the surface coil used for signal detection are a composite of the oxygendependent line widths from each of the ps in reality because of the relatively broad lines from the india ink ps the range of oxygen levels that are likely to be present in the tissue and the limited number of ps in each subregion it is a challenge to resolve directly even the major groups of similar line widths therefore using the observed line width to provide a quantitative measure of oxygen would seem to have modest utility in itselfthe other method of clinical epr oximetry is based on the use of microcrystalline probes eg lipc lincbuo encapsulated in biocompatible polymers swartz et a0al a0 clinical measurements currently are being performed using the oxychip which consists of oxygen sensitive microcrystals of lithium octanbutoxynaphthalocyanine lincbuo embedded in polydimethylsiloxane pdms hou khan gohain kuppusamy kuppusamy a0 hou et a0al a0 jarvis et a0al a0 the dimensions currently used in humans are cylinders that are a0 mm long with a diameter of a0mm the epr signal from the sensor oxychip reflects the po2 within the pdms which itself reflects an average of the po2 in contact with the external surface of the cylinder the dimensions of the oxy | 0 |
immunerelated genes pairs signature predict the prognosis of cervical cancer patientsHan Nie1 Fanqin Bu2 Jiasheng Xu1 Taoshen Li1 Jun Huang2To screen the key immune genes in the development of cervical cancer construct immune related gene pairs IRGPs and evaluate their influence on the prognosis of cervical cancer Tumor Genome Atlas TCGA database and geo database were downloaded as training set and validation set respectively and immune related gene data were downloaded from immport IRGPs model is established by machine learning and the model is analyzed and evaluated Using the Uclcan to analyze the immune genes expression in cervical cancer and to further explore the association with the expression level and the clinical stage and prognosis of cervical cancer According to the analysis of training set we identified IRGPs as key gene pairs and constructed the model The AUC value of the model was greater than and the model group survival rate was conspicuous different P The reliability of the model was confirmed in the validation group Our IRGPs play an important role in the occurrence and development of cervical cancer and can be used as a prognostic marker and potential new target of cervical cancerCervical cancer is one of the four most common gynecological tumors1 Every year at least women in the world are diagnosed with cervical cancer and more than people are killed2 In recent years the incidence rate of cervical cancer has decreased significantly through universal screening and health knowledge However the incidence rate of cervical cancer is still high in developing countries3 For women with low education in less developed areas the coverage rate of cervical cancer screening is still very low4 Squamous cell carcinoma is the most common type of cervical cancer accounting for of cervical cancer cases while adenocarcinoma only accounts for about In developing countries of cervical cancer patients have local infiltration or metastasis which has led to the high mortality of cervical cancer in developing countries Early cervical cancer is usually treated by radical hysterectomy When there are risk factors such as lymph node metastasis and endometriosis that may lead to recurrence they will be treated with chemotherapy5 The standard treatment for patients with locally advanced cervical cancer is conventional radiation therapyCRT6 The fiveyear survival rate of patients with locally advanced cervical cancer can be as high as after surgical resection radiotherapy chemotherapy CRT and so on7 However at present all treatment methods are not effective for patients with paraaortic lymph node metastasis and their threeyear progression free survival time PFS and total survival time OS are and respectively The fiveyear survival rate of cervical cancer patients with recurrence and metastasis was as low as Limited treatment is the main reason for this situation Now palliative chemotherapy is the most commonly used for patients with metastatic and recurrent cervical cancer10 The median survival time of patients with metastatic or recurrent cervical cancer treated with platinumtaxane chemotherapy and bevacizumab can be extended to a0months11 However these treatments are far from enough for most locally advanced and metastatic cervical cancer patients with positive lymph node metastasisIn recent years immunotherapy has been developed and increasingly used in cancer patients For example PDL1 is overexpressed in a variety of tumor cells including liver cancer cells and lung cancer cells and plays an important role in regulating the immune response of tumor cells12 Currently there are several clinical trials involving FDAapproved immunosuppressive checkpoint inhibitors which attack tumor cells expressing PDL1 by blocking the PDL1PD1 signaling pathway so as to improve the treatment and prognosis of patients From the current situation immunosuppressive therapy has achieved good results in many solid tumors16 The 1Department of Vascular Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China 2Department of Gastrointestinal Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China email junhuangncu163comScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cresults of PD1PDL1 inhibition in cervical cancer are also satisfactory However at present immunoassay sites with a therapeutic effect are scarce and the research on tumor immunotherapy is far from sufficient In this study we screened immune genes that are significantly related to the prognosis of cervical cancer constructed an immune gene pair IRGP model based on these genes and used it to verify the unique prognostic markers of cervical cancerMethodData acquisition Gene expression profile data of patients with cervical squamous cell carcinoma were obtained from cancer and tumor gene map TCGA wwwtcga and gene expression profile data set gse4400116 was obtained from gene expression compilation GEO wwwncbinlmnihgovgeo database including samples of cervical cancer patientsAcquisition of sample immune gene expression immune related genes were downloaded from immport wwwimmpo rthome including antigen presenting cells chemokines and their receptors cytokines and their receptors interferon interleukin etc Using limma package in R we compared the gene expression data of cervical cancer samples downloaded from TCGA database and geo database as training set and verification set with immune related genes and extracted the expression amount of immune related genes in cervical cancer samplesConstruction of immune related gene pairs IRGPs In the two groups of data processed in the previous step the IRGP of the sample is calculated and the relatively high change is selected according to the standard of media absolute deviation IRGP values are calculated by comparing gene expression levels in specific samples or profiles in pairs The immune related genes are matched to compare the IRGPs If the first IRG is larger than the second IRG the output of the IRGP is otherwise the output is If the ratio of IRGP score of or in training set and verification set is higher than then remove the IRGP and retain the remaining IRGP as candidate IRGP for prognosis prediction The logistic rank test was used to screen the prognosis IRGP FDR Cox risk regression analysis and glment in R were used to perform tenfold cross validation to analyze the candidate IRGP and obtain the IRGP index We constructed the best gene pairs as immune gene pair model We use ROC to calculate the optimal cutoff value of IRGP index and use it as the basis to distinguish high and low risk groupsIRGPs model validation The single factor and multi factor Cox proportional risk analysis and survival analysis of TCGA and gse44001 cervical cancer samples were carried out with IRGPs modelInfiltration of immune cells in cervical cancer samples In order to study the infiltration of immune cells in the high and low risk groups of cervical cancer we used CIBERSORT17 to evaluate and predict the enrichment of immune cells in the samples CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression RNA SEQ data can be used to estimate the infiltration of immune cells CIBERSORT can analyze the relative abundance of immune infiltrating cells in each sample including NK cells T cells B cells and macrophagesFunctional enrichment analysis of GSEA go and KEGG Gene set enrichment analysisGSEA enrichment analysis was carried out for each gene related to immune prognosis using the fgsea package in R Cluster profiler19 was used to enrich Gene ontologyGOfunction and KEGG pathway Significant enrichment criteria the absolute value of NES is greater than the nomp value is less than and the fdrq value is less than Expression of immune gene in cervical cancer Ualcan were used to analyze the expression of immune genes in cervical cancerStatistical analysis Measured data were expressed as mean ± standard deviation x ± s and data were compared using t test Kaplan Meier method was used for survival analysis The receiver operating characteristic curve ROC curve and ROC analysis were completed by survivalROC103 Single factor and multi factor analysis using Cox proportional risk regression model P was statistically significant P a0 as the difference has very significant statistical significanceEthical approval and consent to participate This article does not contain any studies with patients or animals performed by any of the authorsResultsExpression of immune related genes and construction of IRGPs in cervical cancer samples We obtained gene expression data of cervical cancer samples from TCGA database as training set cervical cancer samples from gse44001 as verification set immune related genes from immport and immune related genes from cervical cancer samples by comparing the two Through these immune related genes we constructed IRGPs We remove more than of the IRGP with a score of or from the training set and validation set leaving IRGP as candidates Combine TCGA clinical data with training set data Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cTCGA clincial dataAge ¥ ¥ GradeG1G2G3G4TT1T2T3T4MM0M1NN0N1Table TCGA clinical dataIRG1APOBEC3HARG2BTCCCL2CCL20CCL20CCL20CCL28CXCL1CXCL2DESDESDLL4FLT3LGHCKHCKHLADQA2IL1BIL1BJAK1NOD1NRP1PLXNB3PSMD7RBP7RBP7STC1TLR3VAV3Immune processesAntimicrobialsAntimicrobialsCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesAntimicrobialsCytokinesAntimicrobialsAntimicrobialsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsAntimicrobialsAntimicrobialsCytokine_ReceptorsCytokine_ReceptorsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsCytokinesAntimicrobialsBCRSignalingPathwayTable Model information about IRGPIImmune processesCytokinesCytokinesCytokinesCytokine_ReceptorsIRG2BTCCLCF1IL16FGFR3APOBEC3C AntimicrobialsARAFPLXNA1MAP3K14TNFSF10PTAFREPORVEGFCDESINHBASAA2STC2LTB4R2DUOX1EDN1APOBEC3C AntimicrobialsCSF2RBCD3DFGFR2SHC1CXCR3DESTNFRSF18CXCR6NRP1NaturalKiller_Cell_CytotoxicityChemokine_ReceptorsTCRsignalingPathwayTNF_Family_MembersChemokine_ReceptorsCytokine_ReceptorsCytokinesCytokinesTGFb_Family_MemberChemokinesCytokinesCytokine_ReceptorsAntimicrobialsChemokinesCytokine_ReceptorsTCRsignalingPathwayCytokine_ReceptorsNaturalKiller_Cell_CytotoxicityChemokine_ReceptorsCytokinesCytokine_ReceptorsAntimicrobialsCytokine_ReceptorsCoefficientScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A Timedependent ROC curve for IRGPI in the training cohort B Timedependent ROC curve for IRGPI in a0year C Timedependent ROC curve for IRGPI in a0year D Timedependent ROC curve for IRGPI in a0yearTable a0 prognosis related IRGPs were screened by lasso Cox proportional risk regression analysis After iterations we selected optimal IRGPs to build the immune prognosis model Table a0IRGPs model validation The immune prognosis model was applied to the training set and the patients in each training set were scored According to ROC curve analysis the optimal cutoff value for patients to be divided into high and low risk groups is Fig a01A After evaluating the model we found that AUC value of model and a0years is Fig a01B Fig a01C and Fig a01D The results show that our immune prognosis gene has a high reliability for the model The training set was divided into highrisk group Fig a02A and highrisk group Fig a02B The results showed that the overall survival rate OS of highrisk group was significantly lower than that of lowrisk group For TCGA training set data single factor and multi factor Cox risk regression analysis showed that only IRGPs model showed significant prognostic effect in single factor Cox Fig a02C while age and IRGPs could be significant independent prognostic factors in multi factor Cox Fig a02D Applying this model to the validation set of gse44001 Fig a03A Table a0 survival analysis showed that the OS of patients in the highrisk group was significantly lower than that in the lowrisk group Fig a03B In the single factor and multi factor Cox analysis IRGPs model and tumor size were significantly correlated with prognosis Fig a03CDInfiltration of immune cells in cervical cancer samples Most studies believe that the occurrence and development of tumor are closely related to immune cells so it is an ideal method to study the infiltration of immune cells in tumor We used CIBERSORT to analyze the infiltration of kinds of immune cells in patients with high and low risk groups Figure a04A shows the expression of immune cells in different risk groups Macrophage M0 Fig a04B activated mast cells Fig a04C were significantly overexpressed in the highrisk group while stationary dendritic cells Fig a04D stationary mast cells Fig a04E activated CD4T cells Fig a04F and cd8t cells Fig a04G were overexpressed in the lowrisk groupScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A The model divides the training set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Training set single factor Cox regression analysis forest map D Training set multivariate Cox regression analysis forest mapFunctional enrichment analysis of GSEA go and KEGG We analyzed the function enrichment of IRGP in the model The results of go analysis showed that IRGP in the model was mainly enriched in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the binding of fibroblast growth factors and the activity of tyrosine kinase Fig a05AB KEGG results showed that these IRGP were involved in cytokine cytokine receptor interaction chemokine signaling tumor necrosis factor signaling MAPK signaling NF kappa B signaling natural killer cellmediated cytotoxicity viral proteins and cytokines and Th1 and Th2 cell differentiation Fig a05CD The results of GSEA Fig a06A showed that these IRGP were significantly enriched in trace ribonucleoprotein complex Fig a06B neurotransmitter transporter activity Fig a06C endopeptidase activity Fig a06D fibroblast growth factor receptor binding Fig a06E hormone activity Fig a06F fibroblast cell proliferation Fig a06G and growth factor receptor binding Fig a06HExpression of immune gene in cervical cancer We explored the expression of IRGP in cervical cancer using the ualcan model Table a0 There were lowlevel expression of IRGP in cervical cancer Fig a0 and highlevel expression of IRGP Fig a0 There were differences in the expression of low expression IRGP and high expression IRGP in different age groups Fig a0 and there were differences in the expression of IRGP in different stages of cervical cancer Fig a0DiscussionCervical cancer is one of the most common gynecological malignancies HPV infection is considered to be the main cause of cervical cancer2122 although the incidence rate of cervical cancer has been significantly decreased due to the development and promotion of HPV vaccine23 But incidence rate of cervical cancer is still high in developing countries and Chinas low income countries24 At present for cervical cancer patients without invasion and lymphatic metastasis the effect of surgery combined with radiotherapy and chemotherapy is better If metastasis and infiltration occur the treatment effect of cervical cancer patients will become very unsatisfactory In recent years immunotherapy has performed well in a variety of cancers including cervical cancer25 Blocking PDL1 PD1 signaling pathway to attack tumor cells expressing PDL1 is the current mainstream method28 Although the anticancer activity of PD1 and PDL1 inhibitors is exciting such immunotherapy is not effective for all patients and a metaanalysis shows that patients who receive PD1 PDL1 inhibitors have a Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A The model divides the validation set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Validation set single factor Cox regression analysis forest map D Validation set multivariate Cox regression analysis forest mapGSE44001 clincial dataStageLargest diameter cm ¥ ¥ ¥ Table GSE44001 clincial datahigher risk of rash thyroid dysfunction pruritus pneumonia and colitis29 Therefore it is of great significance for the detection and treatment of cervical cancer to predict and find more biomarkers that may be related to immune prognosisAt present most of the prognostic genes need to be standardized to reduce the errors caused by sequencing platform and samples In this study the scores of IRGPs constructed by us are calculated from the gene expression data of the same sample which can not only ignore the impact of different platforms but also do not need to standardize and scale the data This method has been used in many studies including cancer molecular classification with high reliability3233In this study we screened pairs of IRGP to construct the immune prognosis model related to the overall survival rate of cervical cancer patients The AUC values of the model in and a0years were all greater than According to these pairs of IRGP they were divided into highrisk group and lowrisk group In TCGA training group and GSE44001 verification group the OS of highrisk group was significantly lower than that of lowrisk group P These pairs of IRGP have a good effect on sample discrimination We found that macrophage Mo and activated mast cells were significantly over expressed in highrisk group by immunocyte infiltration analysis of samples The existing research shows that mast cells and macrophages play an important Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Immune infiltration status within IRGPI risk groups B Expression of Macrophage M0 C Expression of Mast cells activated D Expression of Dendritic cells resting E Expression of Mast cells resting F Expression of T cells CD4 memory activated G T cells CD8role in cervical cancer which can promote the development of cervical cancer by promoting lymphangiogenesis and angiogenesis34 However in the lowrisk group the expression of static dendritic cells static mast cells activated CD4T cells and cd8t cells is high Although the effect of CD4T cells on cervical cancer has not been agreed the cd8t cells are closely related to the better prognosis of cervical cancer patients37 there is evidence that dendritic cells will decrease in patients with high HPV infection which indicates that high expression of dendritic cells is beneficial to resist cervical cancer40 which is consistent with our results The enrichment analysis of go and GSEA showed that these immune genes were mainly involved in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the activity of metalloendopeptidase the binding of fibroblast growth factors and their receptors hormone activity fibroblast proliferation and the binding process of growth Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Histogram graph of Immunerelated genes GO analysis results B Point graph of Immunerelated genes GO analysis results C Histogram graph of Immunerelated genes KEGG pathway analysis results D Point graph of Immunerelated genes KEGG pathway analysis resultsfactor receptorsAs we all know cytokines and chemokines are the key factors in the immune response for example In cervical cancer IL10 can interfere with the differentiation of dendritic cells and thus play a strong immunosuppressive effectTGFβ can inhibit T cell proliferation and attenuate immune response41 Research shows that growth factors and epidermal growth factors are closely related to the growth of cervical cancer and the survival rate of cervical cancer patients High expression of growth factors and epidermal growth factors often predict poor prognosis42 Growth of fibroblasts can stimulate angiogenesis at the early stage of tumor The proliferation and invasion of cancer cells and the remodeling of extracellular matrix promote the growth of cervical cancer4546 KEGG results showed that these immune genes were mainly enriched in chemokine signaling pathway tumor necrosis factor signaling pathway MAPK signaling pathway NF kappa B signaling pathway natural killer cellmediated cytotoxicity viral protein and cytokine and Th1 and Th2 cell differentiation Th1 and Th2 may be involved in the pathogenesis and growth of cervical cancer Th1 may be the target of predicting chemotherapy response of advanced cervical cancer47 while other pathways are classical signal pathways related to cancer Immune cytokines play an important role in cervical lesions Torres et a0al Found that IL10 is highly expressed in the cervix of women with persistent HPV which may be related to the persistence of HPV and the promotion of disease progression Further research by their team showed that copy individuals of IL4 IL6 IL10 and TGFB1 were significantly associated with cervical cancer and could be used as biomarkers for susceptibility to the disease5152These pairs of IRGP have different immune genes most of which are cytokines antimicrobial agents and natural killer cells which are involved in various stimulation reactions and play a key role In cervical cancer HPV can inhibit the apoptosis of cervical cancer cells by down regulating NOD153 In our sample we also found that the expression of NOD1 in tumor tissue is low and there are differences in different ages and stages Figs a07D 9C 10I Sang Yeon Cho et a0al Found that duox1 is highly expressed in cervical squamous cell carcinoma and can play a good prognostic role by increasing the amount of innate immune cells54 The analysis also showed that DUOX1 is highly expressed in tumor tissues and related to age and grade Figs a08B 9G 10D Stc2 can promote the proliferation of cervical cancer cells and increase the resistance to cisplatin55 while high expression of DDL4 is usually associated with low pelvic lymph node metastasis and survival rate of cervical cancer56 Therefore we believe that the IRGP constructed in this study plays an important role in the development and prognosis of cervical cancerThere are also some deficiencies in our research Although we select data samples from two databases for analysis and use more advanced methods to reduce the errors caused by platforms samples etc this is still a retrospective analysis If we can carry out a prospective study or obtain clinical samples and evaluate them with Western blot or immunohistochemistry it will be more convincingScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A GSEA analysis of immune signature genes BH In the high immune risk group of cervical cancer cancer marker genes were abundant P FDR Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cCLCF1DLL4INHBANOD1NRP1RBP7CXCR3DUOX1FGFR3AgeNormalvsAge4160YrsAge2140YrsvsAge4160YrsAge2140YrsvsAge6180YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsAge6180YrsvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsHLADQA2NormalvsAge4160YrsLTB4R2STC2TNFSF10VAV3NormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge4160YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsAge4160YrsvsAge6180YrsPval777E05123E02483E04115E07209E02433E07270E06354E05281E02982E03260E03384E04322E02163E05600E04868E03106E02109E02122E04396E03281E04459E02421E04876E03441E02105E02384E04566E05218E05803E09139E06225E08111E16204E13725E10305E02422E07693E10174E05433E15162E12550E10341E02803E04159E06458E04192E02524E10415E11559E10162E12162E12934E14108E02208E03StageNormalvsStage2NormalvsStage3Stage1vsStage2Stage1vsStage3Stage1vsStage4NormalvsStage1NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage4Stage3vsStageNormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Pval185E06220E04542E03304E02460E04696E07546E03252E03210E02439E02390E03186E02395E02317E02122E03164E03396E04984E03222E02315E06226E02387E07734E03433E01118E04173E05355E04222E02160E05259E09131E09258E04162E12199E12176E05202E04663E12390E05241E04162E12860E10479E09175E04148E02240E04435E05119E04463E02230E13297E09263E07840E04170E02162E12493E12284E12125E04Table P value of IRGPs expression in different ages and stagesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues B Expression of DLL4 in cervical cancer and normal tissues C Expression of INHBA in cervical cancer and normal tissues D Expression of NOD1 in cervical cancer and normal tissues E Expression of NRP1 in cervical cancer and normal tissues F Expression of RBP7 in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CXCR3 in cervical cancer and normal tissues B Expression of DUOX1 in cervical cancer and normal tissues C Expression of FGFR3 in cervical cancer and normal tissues D Expression of HLADQA2 in cervical cancer and normal tissues E Expression of LTB4R2 in cervical cancer and normal tissues F Expression of STC2 in cervical cancer and normal tissues G Expression of TNFSF10 in cervical cancer and normal tissues H Expression of CESC in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different ages B Expression of DLL4 in cervical cancer and normal tissues at different ages C Expression of NOD1 in cervical cancer and normal tissues at different ages D Expression of NRP1 in cervical cancer and normal tissues at different ages E Expression of RBP7 in cervical cancer and normal tissues at different ages F Expression of CXCR3 in cervical cancer and normal tissues at different ages G Expression of DUOX1 in cervical cancer and normal tissues at different ages H Expression of FGFR3 in cervical cancer and normal tissues at different ages I Expression of HLADQA2 in cervical cancer and normal tissues at different ages J Expression of LTB4R2 in cervical cancer and normal tissues at different ages K Expression of STC2 in cervical cancer and normal tissues at different ages L Expression of TNFSF10 in cervical cancer and normal tissues at different ages M Expression of VAV3 in cervical cancer and normal tissues at different agesConclusionWe constructed an immune gene pair model which is closely related to the prognosis of cervical cancer patients The model contains IRGP and immunerelated genes The biological functions of these immunerelated genes are closely related to the occurrence and development of cervical cancer Therefore we think that these IRGPs may be the target of predicting or diagnosing cervical cancer and suggest that immunotherapy can improve the prognosis of cervical cancer patients by regulating these IRGPsScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different stages B Expression of CXCR3 in cervical cancer and normal tissues at different stages C Expression of DLL4 in cervical cancer and normal tissues at different stages D Expression of DUOX1 in cervical cancer and normal tissues at different stages E Expression of FGFR3 in cervical cancer and normal tissues at different stages F Expression of HLADQA2 in cervical cancer and normal tissues at different stages G Expression of INHBA in cervical cancer and normal tissues at different stages H Expression of LTB4R2 in cervical cancer and normal tissues at different stages I Expression of NOD1 in cervical cancer and normal tissues at different stages J Expression of NRP1 in cervical cancer and normal tissues at different stages K Expression of RBP7 in cervical cancer and normal tissues at different stages L Expression of STC2 in cervical cancer and normal tissues at different stages M Expression of TNFSF10 in cervical cancer and normal tissues at different stages N Expression of VAV3 in cervical cancer and normal tissues at different stagesData availabilityAll data are available Please contact us to access if it is neededReceived May Accepted July References Stewart Bernard W et al Cancer prevention as part of precision medicine plenty to be done Carcinogenesis 101093carci nbgv16 Bray F et al Global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin 103322caac21492 Passos Camila M Sales Jacqueline B Maia Emanuella G Caldeira ThaÃs C M Rodrigues Roberta D Figueiredo N Claro Rafael M Trends in access to female cancer screening in Brazil J Public Health Oxf 101093pubme dfdaa0 Asrabuddhe V V Parham G P Mwanahamuntu M H Vermund S H Cervical cancer prevention in low and middleincome countries feasible affordable essential Cancer Prevent Res 10115819406207CAPR110540 Koh WuiJin AbuRustum Nadeem R Bean Sarah Bradley Kristin Campos Susana M Cho Kathleen R Chon Hye Sook Chu Christina Clark Rachel Cohn David Crispens Marta Ann Damast Shari Dorigo Oliver Eifel Patricia J Fisher Christine M Frederick Peter Gaffney David K Han Ernest Huh Warner K Lurain John R Mariani Andrea Mutch David Nagel Christa Nekhlyudov Larissa Fader Amanda Nickles Remmenga Steven W Reynolds R Kevin Tillmanns Todd Ueda Stefanie Wyse Emily Yashar Catheryn M McMillian Nicole R Scavone Jillian L2019 Cervical Cancer Version NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw 106004jnccn Chen J et al Nanotechnology in the management of cervical cancer Rev Med Virol 25Suppl 101002 Varia M A et al Cervical carcinoma metastatic to paraaortic nodes extended field radiation therapy with concomitant 5fluorouracil and cisplatin chemotherapy a Gynecologic Oncology Group study Int J Radiat Oncol Biol Phys 101016s0360 Randall Leslie M Monk Bradley J Darcy Kathleen M Tian C Burger Robert A Liao SY Peters William A Stock Richard J Fruehauf John P Markers of angiogenesis in highrisk earlystage cervical cancer a Gynecologic Oncology Group study Gynecol Oncol 101016jygyno Boussios S et al Management of patients with recurrentadvanced cervical can | 2 |
High glucose HG induced podocytes injury plays an important role in diabetes nephropathy DN development Long noncoding RNA cancer susceptibility candidate CASC2 was found to be decreased in serum of DN patients We aimed to explore the function and possible mechanism of CASC2 in HG induced podocytes injuryMethods Under normal glucose NG HG and mannitol stimulated podocyte conditions the levels of CASC2 microRNA95p miR95p and peroxisome proliferatoractivated receptor gamma PPARÎ were examined by quantitative realtime polymerase chain reaction qRTPCR Podocyte injury was evaluated by measuring cell viability and apoptosis of CIHP1 cells were checked by cell counting kit8 CCK8 assay and flow cytometry respectively Western blot was used to detect all protein levels Dualluciferase reporter RNA immunoprecipitation RIP and RNA pulldown assays were performed to confirm the relationship between CASC2 and miR95pResults HG stimulation inhibited the expression levels of CASC2 and PPARÎ but promoted the expression of miR95p HG could restrain cell viability autophagy and facilitate apoptosis in CIHP1 cells while CASC2 overexpression could reverse HGinduced podocytes injury Furthermore CASC2 could be used as a ceRNA to adsorb miR95p and miR95p mimic overturned the effects of CASC2 on cell viability autophagy and apoptosis in HGstimulated podocytes Additionally PPARÎ was a target gene of miR95p and CASC2 could weaken the HGinduced podocytes injury by upregulating PPARÎConclusion CASC2 increased cell viability autophagy and inhibited cell apoptosis by regulating miR95pPPARÎ axis thus reducing the HGinduced podocytes injuryKeywords High glucose Podocyte CASC2 miR95p PPARÎCorrespondence gltxs009163com Department of Nephrology Taian Campus of the 960th Hospital of the Chinese Peoples Liberation Army No217 Huanshan Road Taishan District Taian Shandong ChinaFull list of author information is available at the end of the BackgroundDiabetes is a common endocrine disease among which the prevalence of diabetes nephropathy DN is [] It is estimated that the number of DN patients is expected to increase to million by [] DN is characterized by the presence of albuminuria and a decreased glomerular filtration rate [] Podocyte cells podocytes are epithelial cells in the The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLi a0et a0al Diabetol Metab Syndr Page of visceral layer of renal follicles which play a key role in the pathogenesis of DN and are an important component of glomerular filtration barrier [ ] Several studies have revealed the correlation between podocytes injury death and apoptosis and albuminuria [] and reducing podocyte injury can improve DN [] However the mechanism for alleviating podocytes injury remains unclearLong noncoding RNAs lncRNAs are nonproteincoding RNA molecules longer than nucleotides which are widely regarded as the important regulators in cellular function and disease processes [] Increased evidences suggested that lncRNA could modulate DN progression For instance lncRNAs GM5524 and GM15645 could regulate the HGstimulated podocyte autophagy in DN [] LncRNA PVT1 knockdown repressed podocytes injury and apoptosis via increasing FOXA1 [] However there are still many lncRNAs in DN function and molecular mechanisms have not been studiedLncRNA cancer susceptibility candidate CASC2 located on chromosome 10q26 plays a regulatory role as an anticancer factor in various cancers such as hepatocellular carcinoma [] and pancreatic carcinoma [] Recently Wang et a0al revealed that CASC2 was specifically reduced in serum and renal tissues of type diabetes patients with chronic renal failure and followup identified that the serum of patients with low CASC2 expression had higher incidence of chronic renal failure [] MicroRNA95p miR95p is both a tumor depressor and a tumor promoter [ ] A report demonstrated that miR95p was related to complications of nephropathy in Type and Type diabetes patients [] The mechanism by which lncRNA can serve as the competing endogenous RNA ceRNA for miRNA to modulate the abundance of mRNA has been widely reported [] Peroxisome proliferatoractivated receptor gamma PPARÎ is implicated in several metabolic syndromes including DN Downregulated PPARÎ could activate catenin signaling to destroy podocyte architectural integrity and increase cell apoptosis in DN [] Furthermore lncRNA TUG1 could relieve extracellular matrix accumulation by sponging miR377 and regulating PPARÎ in DN [] Based on the above findings we speculated whether CASC2 can modulate PPARÎ expression by serving as a ceRNA of miR95p in DNIn this work we aimed to explore the effects of CASC2 on cell viability apoptosis and autophagy in high glucose HG induced podocytes and probe the relationship among CASC2 miR95p and PPARÎ providing a new perspective on the molecular mechanism of podocytes injury in DNMaterials and a0methodsCell culture and a0high glucose inductionHuman podocytes CIHP1 Ximbio London USA were cultured in a Dulbeccos modified Eagles medium DMEM Invitrogen Carlsbad CA USA containing fetal bovine serum FBS Gibco Carlsbad CA USA at a temperature of a0°C with CO2 When cells density reached about CIHP1 cells were exposed to normal glucose NG a0mM high glucose HG a0mM or mannitol a0mM and the exposure time was determined by individual experiments requiredCell transfectionCASC2 overexpressed plasmid CASC2 and its control Vector small interfering RNAs against CASC2 and PPARÎ siCASC2 siPPARÎ and matched siNC were provided by GenePharma Shanghai China miR95p mimic miR95p inhibitor antimiR95p and their corresponding references miRNC antiNC were synthesized by Beyotime Beijing China Transfection of podocytes was performed by using Lipofectamine InvitrogenQuantitative realtime polymerase chain reaction qRTPCRThe RNA in CIHP1 cells was extracted by TRIzol Invitrogen and the complementary DNA cDNA was synthesized via reverse transcription using HiScript Q RT Super Mix Vazyme Piscataway NJ USA The reverse transcription was performed at a0°C for a0min and at a0°C for a0s qRTPCR analysis was conducted on RealTime PCR System Applied Biosystems Foster City CA USA using the SYBR premix Ex TaqIIkit TaKaRa Wuhan China Glyceraldehyde3phosphate dehydrogenase GAPDH and U6 were used as endogenous controls for CASC2PPARÎ and miR95p respectively The primers used in this paper were synthesized by GenePharma and the sequences were used as below CASC2 forward ²GCA CAT TGG ACG GTG TTT CC3² reverse R ²CCC AGT CCT TCA CAG GTC AC3² miR95p F ²GTG CAG GGT CCG AGGT3² R ²GCG CTC TTT GGT TAT CTA GC3² PPARÎ F ²AGA GCC TTC CAA CTC CCT CA3² R ²AAC AGC TTC TCC TTC TCG GC3² U6 F ²TTG GTG CTC GCT TCG GCA ² R ²GTG CAG GGT CCG AGGT3² GAPDH F ²GGA GTC CAC TGG TGT CTT CA3² R ²GGG AAC TGA GCA ATT GGT GG3²F Cell viability and a0apoptosis detectionCIHP1 cells were tiled into the 96well plates and exposed to different treatments HG NG HG Vector HG CASC2 and so on At given points in time a0h a0h and a0h a0µL cell counting kit8 CCK8 0cLi a0et a0al Diabetol Metab Syndr Page of Beyotime was added to the cells and cultured for another a0h at a0°C Finally the absorbance at a0nm was measured by BiotekEpoch2 Beijing ChinaThe apoptosis of podocytes CIHP1 was estimated at a0h after exposure to different treatments by using an Annexin V fluorescein isothiocyanate FITC and propidium iodide PI apoptosis detection kit Keygen Beijing China Briefly podocytes were collected and were then suspended in a0µL FITC and a0µL PI in the absence of light for a0 min The apoptosis of CIHP1 cells was checked by a flow cytometer BD Biosciences Franklin Lake NJ USAWestern blot assayTotal protein from CIHP1 cells was extracted by RIPA Beyotime and denatured at a0°C for a0min before separation and then transferred to polyvinylidene difluoride PVDF Beyotime membranes Membranes were sealed with milk for a0 h before incubation with primary antibodies against Bcell lymphoma2 BCL2 Abcam Cambridge MA USA Cleavedcaspase3 Abcam Light chain 3II LC3II Abcam LC3I Abcam Beclin Beyotime PPARÎ Abcam or GAPDH Beyotime overnight at a0 °C HRPconjugated secondary antibody Abcam was employed to incubate the membranes for another a0h And the proteins were visualized by using BeyoECL Moon BeyotimeDualluciferase reporter assayCASC2 wild type CASC2wt with miR95p binding sites and its mutant type CASC2mut without binding sites were cotransfected into CIHP1 cells with miR95p or miRNC respectively Transfection was continued for a0h and luciferase activity was evaluated through a Dualluciferase reporter kit Promega Madison WI USA In the same manner PPARÎ ²untranslated region ²UTRwt with miR95p binding sites and PPARÎ ²UTRmut were cotransfected into cells with miR95p or miRNC respectively and the luciferase activity was detectedRNA immunoprecipitation RIP assay and a0RNA pulldown assayRIP detection was conducted using a Magna RIP RNABinding Protein Immunoprecipitation Kit Millipore Billerica MA USA CIHP1 cells were treated with miR95p or miRNC a0h later cells were lysed in RIP Lysis Buffer containing protease inhibitors Then Argonaute2 Ago2 or ImmunoglobulinG IgG antibody Abcam were added to the cell lysates overnight at a0°C and the immunoprecipitated RNAs were obtained CASC2 and miR95p levels were estimated using qRTPCR analysisCIHP1 cells were transfected with Biotin labeled BiomiR95p and BiomiRNC respectively At a0 h postobtained by using a Pierce¢ Magnetic RNA PullDown transfection cells were collected and the bound RNA was Kit Thermo Fisher Scientific Waltham MA USA according to the instructions Finally CASC2 enrichment was assessed by qRTPCRStatistical analysisData were acquired from at least three independent repetitions and displayed as mean ± standard deviation SD Difference analysis was conducted by Students ttest with two groups and oneway analysis of variance ANOVA with multiple groups using GraphPad Prism The P value less than was regarded as statistically distinctResultsCASC2 alleviated the a0HGinduced podocytes injuryFirstly we examined the expression of CASC2 in human podocytes treated with NG HG or mannitol by qRTPCR The results showed that HG significantly decreased CASC2 expression in CIHP1 cells compared with NG and mannitol treatment Fig a0 1a In addition a timedependent reduction in CASC2 expression was displayed in HGtreated CIHP1 cells and a0h Fig a01b In view of the expression of CASC2 was substantially reduced at a0h of HG stimulation we then overexpressed CASC2 in HGstimulated CIHP1 cells for a0 h and overexpression efficiency was identified by qRTPCR As shown in Fig a01c CASC2 expression was obviously promoted in HGstimulated CIHP1 cells after transfection of CASC2 for a0 h CCK8 and flow cytometry results indicated that overexpression of CASC2 induced cell viability Fig a01d and retarded apoptosis Fig a01e in HGtreated CIHP1 cells To confirm the results of apoptosis we detected the expression of apoptosis marker proteins BCL2 and Cleavedcaspase3 Western blot assay demonstrated that upregulation of CASC2 enhanced BCL2 expression and silenced Cleavedcaspase3 expression Fig a0 1f which was in agreement with the results of Annexin VFITCPI Furthermore HG could reduce the ratio of LC3IILC3I and Beclin expression in CIHP1 cells and CASC2 overexpression reversed the effects of HG on the expression of autophagy related proteins Fig a01g The above findings indicated that CASC2 could alleviate the HGinduced podocytes injury by affecting cell viability apoptosis and autophagyCASC2 directly interacted with a0miR5pLncRNA generally functions as a sponge for miRNA in human diseases [] We speculated whether CASC2 could also act as miRNA sponge to regulate 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 alleviated the HGinduced podocytes injury a The expression of CASC2in CIHP1 cells treated with normal glucose NG high glucose HG or mannitol was detected by qRTPCR b After CIHP1 cells were treated with HG mM for h h and h respectively CASC2 expression was measured by qRTPCR c CIHP1 cells were divided into four groups which were control NG mM HG mM HG vector and HG CASC2 CASC2 expression was detected by qRTPCR d Cell viability was assessed by CCK8 assay e Cell apoptosis was examined by flow cytometry f g Western blot assay was used to determine the expression levels of apoptosisrelated proteins BCL2 and Cleavedcaspase3 and autophagy related proteins LC3II LC3I and Beclin P 0cLi a0et a0al Diabetol Metab Syndr Page of HGinduced podocytes injury As shown in Fig a0 2a we found that miR95p was upregulated in HGtreated CIHP1 cells compared to cells treated with NG or mannitol and miR95p expression was drastically augmented in HGtreated CIHP1 cells in a timedependent manner Fig a0 2b Interestingly there were complementary sites between miR95p and CASC2 by bioinformatics website starBase v20 Fig a0 2c Dualluciferase reporter assay showed that the luciferase activity of CASC2wt was obviously decreased in CIHP1 cells transfected with miR95p than that cells transfected with miRNC whereas it was no significant difference in luciferase activity of CASC2mut Fig a02d RIP assay indicated that the enrichments of CASC2 and miR95p were higher in CIHP1 cells incubated with Ago2 Fig a02e RNA pulldown assay further revealed that the enrichment of CASC2 in BiomiR95p group was aggrandized relative to that BioNC group Fig a02f These results strongly supported that CASC2 could specifically bind to miR95p Meanwhile qRTPCR data showed that CASC2 knockdown in CIHP1 cells elevated miR95p expression and CASC2 overexpression degraded miR95p expression Fig a02g h These results suggested that CASC2 could act as a ceRNA to negatively regulated miR95p expression in podocytesCASC2 regulated the a0HGinduced podocytes injury via a0targeting miR5pAs presented in Fig a0 3a miR95p mimic miR95p could reverse the inhibitory effect of CASC2 overexpression on miR95p expression in HGinduced CIHP1 cells As expected the impact of CASC2 on promoting cell activity Fig a03b and inhibiting cell apoptosis Fig a03c in HGstimulated CIHP1 cells was offset by miR95p Simultaneously the inhibition of CASC2 on the protein expression of Cleavedcaspase3 and the promotion of CASC2 on LC3IILC3I ratio as well as the levels of BCL2 and Beclin could be weakened by transfection of miR95p in HGinduced CIHP1 cells Fig a03d e The obtained data proved that CASC2 attenuated the HGinduced podocytes injury by downregulating miR95pFig CASC2 directly interacted with miR95p a The expression of miR95pin CIHP1 cells treated with normal glucose NG high glucose HG or mannitol was measured by qRTPCR b After CIHP1 cells were treated with HG mM for h h and h respectively miR95p expression was examined by qRTPCR c StarBase v20 was used to predict the target miRNAs of CASC2 df Dual luciferase reporter RIP and RNA pulldown assays were utilized to assess the combination of CASC2 and miR95p g CASC2 expression in CIHP1 cells transfected with siNC or siCASC2 was determined by qRTPCR h The expression of miR95pin CIHP1 cells transfected with siNC siCASC2 Vector or CASC2 was measured using qRTPCR analysis P 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 regulated the HGinduced podocytes injury via targeting miR95p The HGtreated CIHP1 cells were divided into four groups Vector CASC2 CASC2 miRNC and HG miR95p a The expression of miR95p was examined by qRTPCR b c Cell viability and apoptosis were evaluated by CCK8 assay and flow cytometry respectively d e The expression levels of apoptosisrelated proteins BCL2 and Cleavedcaspase3 and autophagy related proteins LC3II LC3I and Beclin were detected by western blot assay P CASC2 acted as a0a a0ceRNA by a0sponging miR5p to a0facilitate PPARÎ expressionAs appeared in Fig a04ad HG inhibited the mRNA and protein levels of PPARÎ in CIHP1 cells compared to NG and mannitol stimulation At a0 h after the induction of HG the mRNA and protein levels of PPARÎ were dwindled in CIHP1 cells The effect of HG treatment on PPARÎ expression was the opposite of that of miR95p thus we speculated whether there was a connection between miR95p and PPARÎ As presented in Fig a04e there were binding sites for miR95p in the ²UTR of PPARÎ Dualluciferase reporter assay showed that miR95p markedly decreased the luciferase activity of PPARÎ ²UTRwt in CIHP1 cells than that PPARÎ ²UTRmut Fig a0 4f suggesting PPARÎ was the target mRNA of miR95p Then we examined the effect of miR95p on PPARÎ expression the interference efficiency of antimiR95p on miR95p expression was first examined by qRTPCR Fig a0 4g Western blot data showed that the overexpressed miR95p could restrain the protein expression of PPARÎ while the decreased miR95p could raise PPARÎ protein expression Fig a04h Additionally we found that CASC2 depletion reduced the protein expression of PPARÎ and cotransfection of antimiR95p could reverse this effect Fig a04i The above findings revealed that CASC2 positively regulated PPARÎ expression by acting as a ceRNA for miR95p in podocytesCASC2 alleviated the a0HGinduced podocytes injury by a0increasing PPARÎConsidering CASC2 could act as a sponge of miR95p to regulate the expression of PPARÎ we further investigated whether PPARÎ was involved in regulation of HGinduced podocytes injury mediated by CASC2 Western blot results indicated that cotransfection of siPPARÎ neutralized the promoting effect of CASC2 on PPARÎ protein expression Fig a0 5a The data of CCK8 and Annexin VFITCPI assays indicated that the effects of CASC2 on cell viability Fig a0 5b and apoptosis Fig a0 5c could be abolished by silencing 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 acted as a ceRNA by sponging miR95p to facilitate PPARÎ expression a The mRNA expression of PPARÎ in NG HG or mannitoltreated CIHP1 cells was analyzed by qRTPCR b qRTPCR assay was used to measure the mRNA expression of PPARÎ in CIHP1 cells treated by HG mM at different times c The protein expression of PPARÎ in NG HG or mannitoltreated CIHP1 cells was analyzed by western blot assay d Western blot assay was used to measure the protein expression of PPARÎ in CIHP1 cells treated by HG mM at different times e StarBase v20 predicted that there were binding sites between miR95p and PPARÎ f Dual luciferase reporter assay was conducted to detect the interaction between miR95p and PPARÎ in CIHP1 cells g The expression of miR95p in CIHP1 cells transfected with antiNC or antimiR95p was measured by qRTPCR h The protein expression of PPARÎ in CIHP1 cells transfected with miRNC miR95p antiNC or antimiR95p was assessed using western blot assay i PPARÎ protein expression in CIHP1 cells transfected with siNC siCASC2 siCASC2 antiNC or siCASC2 antimiR95p was estimated by western blot P PPARÎ in HGinduced CIHP1 cells Similarly the effects of CASC2 on levels of Cleavedcaspase3 BCL Beclin and LC3IILC3I ratio were rescued by siPPARÎ implying PPARÎ knockdown could increase Cleavedcaspase3 protein and decrease the expression levels of BCL2 and Beclin as well as the ratio of LC3IILC3I Fig a05d e To sum up CASC2 alleviated the HGinduced podocytes injury by upregulating PPARÎOverall it could be concluded that HG inhibited cell viability autophagy but promoted cell apoptosis by downregulating CASC2 and PPARÎ expression as well as upregulating miR95p in CIHP1 cells Fig a0DiscussionPodocytes are terminally differentiated visceral epithelial cells which are important components of the glomerular filtration barrier Podocyte viability and apoptosis as well as autophagy can affect glomerular function [] A large number of studies have shown that high glucose induction can cause podocytes injury []Several lncRNAs such as lncRNA MALAT1 [] and lncRNA PRINS [] have been found to be involved in the development of DN they regulated mRNA expression at the posttranscriptional level In this study we found that CASC2 expression was prominently downregulated in high glucosestimulated podocytes in a 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 alleviated the HGinduced podocytes injury by increasing PPARÎ The HGtreated CIHP1 cells were transfected with Vector CASC2 CASC2 siNC and siPPARÎ respectively a PPARÎ protein expression was examined by western blot b c Cell viability and apoptosis were determined by CCK8 assay and Flow cytometry respectively d e The expression levels of BCL2 Cleavedcaspase3 LC3II LC3I and Beclin were checked by western blot assay P timedependent manner and doseresponse manner Autophagy is a doubleedged sword and its excessive activation or repression can cause podocytes injury [] Autophagy activity is impaired in DN patients so promoting autophagy to some extent can reduce podocytes injury [] and Beclin LC3I and LC3II have been shown to be autophagy specific proteins [] In accordance with previous data high glucose could inhibit cell viability and autophagy and promote cell apoptosis while overexpression of CASC2 could attenuate the effect of high glucose on podocytes injury suggesting the protective effect of CASC2 on podocytes injury Similarly Yang et a0al observed that CASC2 was enormously decreased in DN patients while there was no significant difference in CASC2 expression in DN patients as compared to those with DM without complication DN [] Besides Wang et a0 al reported that the development of type diabetes might have no significant effects on CASC2 expression in renal tissue whereas CASC2 expression in renal tissues was found to be evidently lower in patients with type diabetes complicated with chronic renal failure [] These data suggested that 0cLi a0et a0al Diabetol Metab Syndr Page of Fig Schema presented the mechanism that HG repressed cell viability autophagy and promoted cell apoptosis by regulating the CASC2miR95pPPARÎ axis in CIHP1 cellsCASC2 inhibition was very likely to be involved in the pathogenesis of DN Moreover lncRNA often functions as ceRNA and we speculated that CASC2 might also be involved in the regulation of podocytes injury by sponging miRNACompared with nondiabetic subjects the level of miR95p was higher in serum of patients with gestational diabetes mellitus [] and serum miR9 might be an underlying marker for poor prognosis of DN [] In our data the abundance of miR95p was increased in high glucoseinduced podocytes and miR95p was validated to be the target miRNA for CASC2 and CASC2 could inversely modulate miR95p expression in podocytes Recovery experiments showed that CASC2 mitigated podocytes injury by decreasing miR95p via acting as a miR95p sponge similar to the work of Zhang et a0al who indicated that lncRNA SOX2OT could reduce the high glucosestimulated podocytes damage by autophagy induction through binding to miR9 [] Therefore it is reasonable to infer that CASC2 regulated podocyte activity apoptosis and autophagy through sponging miR95pPPARÎ agonists have been widely reported to improve glycemic status in diabetes patients [] and PPARÎ has favorable renal protective effects [] As expected high glucose treatment obviously retarded PPARÎ expression Importantly miR95p directly targeted PPARÎ ²UTR and negatively modulated its expression In addition CASC2 could regulate PPARÎ expression by sponging miR95p based on these results we hypothesized whether CASC2 implicated in podocytes injury by regulating PPARÎ The results showed that PPARÎ knockdown neutralized the effect of CASC2 on podocytes injury Besides PPARÎ has been shown to restore podocyte integrity to improve proteinuria []ConclusionIn summary we believed that CASC2 mainly upregulated the expression of PPARÎ by acting as the ceRNA of miR95p thus alleviating HGinduced podocytes injury through increasing cell viability autophagy and reducing cell apoptosis This study provided a new molecular regulatory mechanism for podocytes injury induced by HG in DNAbbreviationsHG High glucose DN Diabetes nephropathy CASC2 Cancer susceptibility candidate NG Normal glucose RIP RNA immunoprecipitation PPARÎ Peroxisome proliferatoractivated receptor gammaAcknowledgementsThe authors sincerely appreciate all members participated in this study Authors contributionsFL designed the experiments performed the experiments and analyzed and collected the data wrote the manuscript BD analyzed interpreted the data performed the experiments and wrote the manuscript XN performed the experiments and analyzed the data All authors read and approved the final manuscript FundingThis work was supported by Key Research Development Program of Shandong Province China [Grant No2019GSF108172] Natural Science Foundation of Shandong Province China [Grant No2016ZRA08005] and Science and Technology Development Plans of TCM of Shandong Province China [Grant No ] Availability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author on reasonable request 0cLi a0et a0al Diabetol Metab Syndr Page of Ethics approval and consent to participateNot applicablePatient consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestAuthor details Department of Nephrology Heze Mudan Peoples Hospital Heze Shandong China Department of Nephrology Liaocheng Peoples Hospital Liaocheng Shandong China Department of Nephrology Taian Campus of the 960th Hospital of the Chinese Peoples Liberation Army No217 Huanshan Road Taishan District Taian Shandong China Received February Accepted July References Dronavalli S Duka I Bakris GL The pathogenesis of diabetic nephropathy Nat Clin Pract Endocrinol Metab Ogurtsova K da Rocha Fernandes JD Huang Y Linnenkamp U Guariguata L Cho NH et al IDF Diabetes Atlas Global estimates for the prevalence of diabetes for and Diabetes Res Clin Pract Afkarian M Zelnick LR Hall YN Heagerty PJ Tuttle K Weiss NS et al Clinical manifestations of kidney disease among US adults with diabetes JAMA Ziyadeh FN Wolf G Pathogenesis of the podocytopathy and proteinuria in diabetic glomerulopathy Curr Diabetes Rev Berthier CC Zhang H Schin M Henger A Nelson RG Yee B et al Enhanced expression of Janus kinasesignal transducer and activator of transcription pathway members in human diabetic nephropathy Diabetes White KE Bilous RW Marshall SM El Nahas M Remuzzi G Piras G et al Podocyte number in normotensive type diabetic patients with albuminuria Diabetes Mathieson PW The podocyte as a target for therapiesnew and old Nat Rev Nephrol Boon RA Jae N Holdt L Dimmeler S Long noncoding RNAs from clinical genetics to therapeutic targets J Am Coll Cardiol Feng Y Chen S Xu J Zhu Q Ye X Ding D et al Dysregulation of lncRNAs GM5524 and GM15645 involved in high glucose induced podocyte apoptosis and autophagy in diabetic nephropathy Mol Med Rep Liu DW Zhang JH Liu FX Wang XT Pan SK Jiang DK et al Silencing of long noncoding RNA PVT1 inhibits podocyte damage and apoptosis in diabetic nephropathy by upregulating FOXA1 Exp Mol Med Refai NS Louka ML Halim HY Montasser I Long noncoding RNAs CASC2 and TUG1 in hepatocellular carcinoma Clinical significance J Gene Med 2019219e3112 Zhang H Feng X Zhang M Liu A Tian L Bo W et al Long noncoding RNA CASC2 upregulates PTEN to suppress pancreatic carcinoma cell metastasis by downregulating miR21 Cancer Cell Int Wang L Su N Zhang Y Wang G Clinical significance of serum lncRNA cancer susceptibility candidate CASC2 for chronic renal failure in patients with type diabetes Med Sci Monit Fan Y Shi Y Lin Z Huang X Li J Huang W et al miR95p suppresses malignant biological behaviors of human gastric cancer cells by negative regulation of TNFAIP8L3 Dig Dis Sci Wu M Huang Y Chen T Wang W Yang S Ye Z et al LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR95pQKI5 axis J Cell Mol Med Massaro JD Polli CD Costa ESM Alves CC Passos GA SakamotoHojo ET et al Posttranscriptional markers associated with clinical complications in Type and Type diabetes mellitus Mol Cell Endocrinol Zhang J Liu L Li J Le TD LncmiRSRN identification and analysis of long noncoding RNA related miRNA sponge regulatory network in human cancer Bioinformatics Zhou 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Acute myeloid leukemia AML is a complex hematological disease characterized by genetic and clinical heterogeneity The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients Compared with recurrent chromosomal translocations in AML t816p112p133 can be found in any age group but is very rare and typically associated with poor prognosisMethods Conventional cytogenetic studies were performed among AML patients recorded in our oncology database over the last years Fluorescence in situ hybridization FISH was carried out to detect the translocation fusion Array comparative genome hybridization aCGH was carried out to further characterize the duplication of chromosomesResults We identified three AML patients with t816p112p133 by chromosome analysis Two of the three patients who harbored an additional 1q duplication were detected by FISH and aCGH aCGH characterized a Mb and Mb gain in chromosome at band q321q44 separately in these two patients One patient achieved complete remission CR but relapsed months later The other patient never experienced CR and died years after diagnosisConclusion A 1q duplication was detected in two of three AML patients with t816p112p133 suggesting that 1q duplication can be a recurrent event in AML patients with t816 In concert with the findings of previous studies on similar patients our work suggests that 1q duplication may also be an unfavorable prognostic factor of the diseaseKeywords 1q duplication Acute myeloid leukemia t816p112p133 Prognostic factorBackgroundAcute myeloid leukemia AML is a common disease characterized by immature myeloid cell proliferation and bone marrow failure which can be subdivided into pathogenetically different subtypes [] Over the past two decades the incidence has increased by [ ] Furthermore AML has poor longterm survival with a Correspondence lzhang202003163com Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of ChinaFull list of author information is available at the end of the high relapse rate [] Therefore AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies The development of newer effective treatment strategies is necessary for AML patientsTo date the detection of cytogenetic abnormalities has been regarded as a critical prognostic tool for AML treatment [] Hence it is urgently necessary to identify chromosomal rearrangements in AML patients and provide the whole spectrum of cytogenetic abnormalities for AML [] According to the World Health anization classification system updated in AML with recurrent genetic abnormalities including t821q22q22 The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLiu a0et a0al Mol Cytogenet Page of t1517q24q21 t1517PMLRARA t11q23MLL inv16p131q22 and t1616p131q22 has been identified [ ] Nonrandom chromosomal abnormalities such as deletions and translocations have been detected in approximately of all adult AML patients Moreover chromosomal abnormalities have been recognized as genetic events that can cause and promote this disease [] Certain cytogenetic abnormalities including t821q22q22 t1517q24q21 and inv16p131q22 are associated with longer remission and survival while alterations of chromosomes 11q23 and complex karyotypes are associated with poor response to therapy and shorter overall survival [] Chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBMYH11 and t11q23MLL are usually found in AML patients [ ] However AML with t816p112p133KAT6ACREBBP is a very rare AML subtype and can be found in any age group from infancy to the eighth decade of life with a female predominance [] A majority of adult patients with t816p112p133 are therapy related [] and pediatric patients tend to be de novo [] There are approximately cases reported in the literature [] and the first t816p112p133 in an infant was described in [] Some AML patients with t816 p112p133 have a bleeding tendency and disseminated intravascular coagulopathy which are overlapping clinical features that mimic acute promyelocytic leukemia APL [] Unlike APL AML with t816p112p133 has an unfavorable treatment response and outcome [ ] As a sole chromosomal anomaly t816p112p133 is found in more than of reported cases and one or more additional chromosomal anomalies can be seen in the remaining cases [] The most common secondary chromosomal anomalies are total or partial trisomy and monosomy or deletion of the long or short arm of chromosome [ ] Comparatively the gain of 1q in variable sizes has also been frequently noticed in patients with t816p112p133 in these large studies [ ]Recurrent cytogenetic abnormality t816p112p133 is seldom associated with AML and the 1q duplication in AML patients with t816p112p133 has never been discussed In the present study a total of de novo or treatmentrelated AML patients were collected from our laboratory oncology database Among them three patients were detected with t816p112p133KAT6ACREBBP and two of these three showed an additional copy of partial chromosome 1qMethodsPatientsThis study was approved by the Institutional Review Board IRB of Oklahoma University IRB Number A total of AML patient samples were studied cytogenetically from to at the Genetics Laboratory of Oklahoma University Health Sciences Center Bone marrow samples were obtained from three of the patients who had t816p112p133Conventional cytogenetic analysisShortterm cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols Karyotype analysis was performed using Giemsa and trypsin techniques for Gbanding The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature ISCN Fluorescence in a0situ hybridization analysisFluorescence in a0 situ hybridization FISH assays were performed according to the manufacturers instructions in combination with our established laboratory protocols A PMLRARA dualcolor dualfusion translocation probe Abbott Molecular Inc Des Plaines IL USA subtelomerespecific probes for chromosome parm and qarm and whole chromosome painting WCP probes for chromosomes and were purchased from Cytocell Ltd NY USA A spectrum greenlabeled probe mapping to the 8p1121 region and a spectrum orangelabeled probe mapping to the 16p133 region were created in house with the following BACPAC clones RP11642I24[chr8 4167633641856494hg19] and RP11589C21[chr8 4187370242036222hg19] RP11619A23[chr16 37200763914571hg19] and RP1195J11[chr16 38603744025510hg19] Childrens Hospital Oakland Research Institute Oakland CA USA The KAT6A gene located on 8p1121 and the CREBBP gene located on 16p133 were covered by the greenlabeled and redlabeled homebrewed probes respectively All probes were validated before use Chromosome spreads were counterstained with 46diamidino2phenylindole DAPI4 in antifade medium Vector Laboratories Inc CA USA Digital images carrying specific hybridization signals were captured and processed on CytoVision version Applied Spectral Imaging Carlsbad CA USAaCGH analysisGenomic DNA was extracted from each of the three patients bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit Puregene blood kit Qiagen Valencia CA or Nucleic Acid Isolation System QuickGene610L FUJIFILM Corporation Tokyo Japan Two aCGH platforms NimbleGen and Agilent were used in this study For the 0cLiu a0et a0al Mol Cytogenet Page of NimbleGen aCGH platform human reference genomic DNA was purchased from Promega Corporation Promega Corporation Madison WI USA The patients DNA and the reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming and then equal quantities of both labeled products were mixed and loaded onto a a0K oligonucleotide chip Roche NimbleGen Inc Madison WI USA to hybridize at a0 °C for a0 h in a MAUI hybridization system BioMicro Systems Salt Lake City UT according to the manufacturers protocols with minor modifications The slides were washed with washing buffers Roche NimbleGen Inc after hybridization and scanned using a Roche Scanner MS Microarray Scanner Roche NimbleGen Inc Images were analyzed using NimbleScan software version and SignalMap software version Roche NimbleGen Inc The genomic positions were determined using GRCh36hg18 UCSC Genome Browser For the Agilent aCGH platform human reference genomic DNA was purchased from Agilent Corporation Agilent Corporation Santa Clara CA USA The patients DNA and the purchased reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming Agilent Corporation Patient DNA labeled with Cy3 was combined with a normal control DNA sample labeled with Cy5 of the same sex and hybridized to an Agilent à a0K oligo microarray chip Agilent Technologies by incubating in an Agilent Microarray Hybridization Oven Agilent Technologies After a0h of hybridization at a0°C the slides were washed and scanned using the NimbleGen MS Microarray Scanner Roche NimbleGen Inc Agilents CytoGenomics software Agilent Technologies was applied for data analysis The genomic positions were determined using GRCh37hg19 UCSC Genome BrowserCase presentationCase An 82yearold male presented with anemia was referred to us for AML evaluation His subsequent lab results and hospital records were not available in our clinical databaseCase A 28yearold female presented with disseminated intravascular coagulopathy was referred to rule out APL Her complete blood examination and bone marrow aspirate smears were not available Flow cytometry revealed monocytic cells positive for CD4 CD11b partial CD13 bright CD14 partial CD15 CD33 bright and HLADR partial but negative for CD3 CD7 CD34 CD117 MPO and TdT consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient achieved hematological CR on day and cytogenetic CR on day after induction chemotherapy and then relapsed a0months laterCase A 69yearold female with a medical history of breast cancer after lumpectomy chemotherapy and radiation presenting with generalized weakness pancyt ia and fever was referred to us for disease progression evaluation A complete blood examination showed a white blood cell count of à 109L with blasts a hemoglobin count of a0 gL and a platelet count of à 109L Her bone marrow aspirate smear demonstrated over myeloblasts Flow cytometry revealed that of the blast cells expressed CD45 moderate CD34 dim CD38 HLADR CD13 CD15 and CD33 and were negative for CD117 consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient started consolidation chemotherapy but had spontaneous regression and died a0years after AML diagnosisResultsIn case routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes and Fig a01a in of cells consistent with a diagnosis of AML with t816p112p133 The nomenclature of the cytogenetic findings in patient was t816p112p133[]46XY[] No other consistent karyotypic aberrations were detected Thus this male patient was excluded from subsequent FISH and aCGH analysesIn case chromosome analysis demonstrated the same chromosome rearrangement between and in all cells Besides of these cells showed an extra chromosome segment attached to chromosome Fig a01b The karyotypes in patient were described as 46XXt816p112p133 add14p112[]46XY[] Negative FISH t1517q24q21PMLRARA further ruled out a diagnosis of APL data not shown Metaphase FISH analysis confirmed the t816p112p133KAT6ACREBBP fusion and demonstrated a part of chromosome on chromosome Fig a02a and b In addition to characterizing the extrachromosomal material aCGH was carried out aCGH confirmed the FISH findings and detected a a0Mb gain from chromosome at bands q321q44 a0bp GRCh36hg18 USCS Genome Browser Fig a03aIn case t816p112p133 with a gain of a similar chromosome segment on the long arm of chromosome was detected in of cells by karyotyping analysis Fig a0 1c FISH confirmed the KAT6ACREBBP fusion and revealed additional chromosome material Fig a02c and d Loss of the end portion of the chromosome long arm was not found by FISH Fig a03e aCGH further detected a gain from chromosome at bands 1q321q44 results for 0cLiu a0et a0al Mol Cytogenet Page of a Patient b Patient Fig Representative abnormal karyotypes of three patients with t816p112p133 a Karyotype of patient showing 46XYt816p112p133 as the sole abnormality b and c Karyotypes of patients and showing 46XXt816p112p133 and an additional chromosome segment attached to the short arm of chromosome and the long arm of chromosome respectively Translocated derivatives and are indicated by black arrows and derivatives and are indicated by red arrows 0cLiu a0et a0al Mol Cytogenet Page of c Patient Fig continued a0 bp GRCh37hg19 UCSC Genome Browser Fig a0 3b The molecular size was a0MbDiscussionAML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood which accounts for approximately of adult leukemia cases As reported previously common chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBfrequently observed and numerous MYH11 are uncommon chromosomal aberrations also exist in AML [] The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients []t1517PMLRARA and In previous large studies approximately AML cases with t816p112p133 have been reported [] Among them cases showed a gain by 1q of variable sizes [ ] As an uncommon entity t816 accounts for of all cases of AML [] In our study three patients with t816p112p133 were identified one man and two women The two women were both diagnosed with AML subtype M5 and showed an extra copy of 1q at the same bands q321q44 which were different from the nine reported cases above The clinical features and cytogenetic data of the cases of AML with t816p112p133 and 1q duplications are summarized in Table a0 To the best of our knowledge this is the first study of the delineation of 1q duplication by aCGH in AML patients with t816p112p133AML patients with this abnormality often show unique clinical and biological characteristics [] Compared with the current categories t1517 t821 inv16 and t11q23 in AML t816 is clustered closer to t11q23 and shares commonly expressed genes [] Xie et a0 al reported adult AML cases with t816p112p133 indicating that t816p112p133 commonly exhibits monoblastic or myelomonocytic differentiation and arises in patients with a history of cytotoxictreated cancer Patients with de novo AML with t816 or treatmentrelated AML with t816 without adverse prognostic factors have a good outcome [] Identifying adverse prognostic factors is of importance to the choice of therapy and evaluation of survival in AML patients with t816 0cLiu a0et a0al Mol Cytogenet Page of CREBBPKAT6A fusionKAT6A8p1121CREBBPKAT6A fusionKAT6ACREBBP fusionCREBBP16p133CREBBP16p133KAT6ACREBBP fusiona KAT6A8p1121c WCP14WCP1WCP14b WCP1WCP1TelVysion 3q WCP1WCP1WCP3WCP1d WCP3TelVysion 3p TelVysion 3p TelVysion 3q e Fig Metaphase FISH of patient a and c showing KAT6ACREBBP fusion signals WCP FISH indicating the extra chromosomal materials on chromosome and chromosome were both from chromosome b and d No loss of the end portion of the chromosome long arm was indicated eOver the past a0 years cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [] Compared with conventional cytogenetic analysis and FISH methods aCGH is an attractive method for the investigation of cancer genomes [] aCGH has higher resolution simplicity high reproducibility shorter turnaround time and precise mapping of aberrations Most importantly it avoids the need for cell culture and dividing cells [] Furthermore aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [] In our investigation we applied aCGH to characterize the additional chromosome materials in patients and and interestingly found that the two patients 0cLiu a0et a0al Mol Cytogenet Page of revealed the same extra copy of 1q at bands q321q44 Patients with 1q duplication have also demonstrated a wide range of multiple malformations such as intellectual disability macrocephaly large fontanels prominent foreheads broad flat nasal bridges higharched palates retrognathia lowset ears and cardiac defects [ ] More recent studies have shown that a 1q gain is also related to a portion of solid tumors For instance the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [] and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [] In addition patients with a 1q duplication showed worse survival and high risk in acute leukemia Burkitt lymphoma and myeloproliferative neoplasms [] The outcomes of 1q duplication in the nine reported AML patients with t816p112p133 are summarized in Table a0 Seven patients data were available These seven patients two adult and five pediatric all received induction chemotherapy and six achieved CR At the time of last followup two adult patients and three of five pediatric patients had died Only two pediatric patients were alive We reported two adult patients here patient achieved CR but relapsed a0 months later and patient had spontaneous regression and died a0 years after diagnosis Taken together the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t816p112p133 However the significance of the 1q duplication in AML with t816 needs to be further investigated Since such changes have been seldom reported the pathogenic effects of 1q duplication in AML patients with t816p112p133 require more studies to be delineatedConclusionThree patients were detected with t816p112p133 from an AML patient database Two female patients were identified with a 1q duplication by FISH and aCGH analyses Combining our investigation with the findings of published studies we conclude that 1q duplication is a recurrent finding in AML patients with t816 Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AMLFig aCGH results of patient and patient showing partial 1q gain duplicated 1q regions are indicated by red frames 0cLiu a0et a0al Mol Cytogenet Page of Table The previously reported AML cases with a0t816p112p133 and a01q duplicationSex Age years FAB type Karyotype1q BandsOutcome yearsLast stateCase Case FFHaferlach et al FM5M5M5aDiab et alM M4Diab et alDiab et alDiab et alDiab et alFFFFXie et alM Brown et alM Brown et alFM45M4M4M5M4M4M446XXt816p112p133 add14p112[]46XX[]46XXt816p112p133[]46idemadd3q27[]45XXt816p11p13der1013q10q10[]46XXder7t17q21q35t816p11p13[]46XX[]46XY1del1p22t816p11p1310der14t1014q112p112[]47XYdel1q11der1t18p11q112x2i5p10810der14t1014q112p112der16t8165XXt816p11p1318der21t121q12p13[]46XX[]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110q11p11[]46idemadd7p21[]46XX[46XYt816p11p13der14t114q31p11[]46XderXtX1q26q23t816p11p13der11t1111p11q1346XYder3t38q27q13del6p22t816p112p133del10q21q25add13p112del16p12del20p112del20q112q133[]46idemdel1p35p363del15q23add19p131[]46XYt816q27q13del12q21q241del13q21q3116der19t119q32p133mar[]46XYdel6p22t816p112p133[cp2]46XY[]47XderYtY1q12q21 6t816 p11p13[]47idemdel13q3q3 [checked with CAD data]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110 q11p11 []46idemadd7p21 []46XX []1q321q441q321q44CR after inductionRelapsed months laterspontaneous regression1q21NAPartial 1q gain CR for 1q121q111q311q23CR for CR for CR for NA1q32CR for monthsAliveDiedNADeadAliveDiedAliveNADead1q21No CRDied month after treatment1q11Early remission after course Relapsed at months and months after diagnosisDiedAML acute myeloid leukemia FAB FrenchAmericanBritishh M male F female NA not available CR complete remissionAbbreviationsAML Acute myeloid leukemia aCGH Array comparative genomic hybridization FISH Fluorescence in situ hybridization APL Acute promyelocytic leukemia WCP Whole chromosome painting CR Complete remissionAcknowledgementsNot applicableAuthors contributionsM Liu and YR gathered clinical information and drafted the manuscript YR YK and M Liu performed routine cytogenetic analysis and participated in the interpretation of the results M Li performed FISH analysis and participated in the interpretation of the results XL supervised the FISH analysis and helped draft the manuscript XW performed CGH array analysis and helped draft the manuscript LZ and SL conceived the study participated in its design and 0cLiu a0et a0al Mol Cytogenet Page of extensively reviewed and revised the manuscript All authors have read and approved the final manuscriptFundingThis study has received no funding supportAvailability of data and materialsAll data generated or analyzed during this study are included in this published Ethics approval and consent to participateThis study was approved by University of Oklahoma Institutional Review Board for the Protection of Human SubjectsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of China Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City OK USA Department of Neurology The Second Hospital of Jilin University Jilin Peoples Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr Luppi M Fabbiano F Visani G Martinelli G Venditti A Novel agents for acute myeloid leukemia Cancers Basel Cancer Research UK Acute myeloid leukaemia AML incidence statistics https wwwcance rrese archu khealt hprofe ssion alcance rstati stics stati stics bycance rtypeleuka emiaamlincid ence Accessed Aug Jung J Cho BS Kim HJ Han E Jang W Han K Lee JW Chung NG Cho B Kim M Kim Y Reclassification of acute myeloid leukemia according to the WHO classification Ann Lab Med Murphy T Yee KWL Cytarabine and daunorubicin for the treatment of acute myeloid leukemia Expert Opin Pharmacother Byrd JC Mrózek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B CALGB Blood Lindsley RC Mar BG Mazzola E et al Acute myeloid leukemia ontogeny is defined by distinct somatic mutations Blood Vardiman JW Thiele J Arber DA et al The revision of the World Health anization WHO classification of myeloid neoplasms and acute leukemia rationale and important changes Blood Saultz JN Garzon R Acute myeloid leukemia a concise review J Clin Med Döhner H Weisdorf DJ Bloomfield CD Acute myeloid leukemia N Engl J Med Strickland SA Shaver AC Byrne M et al Genotypic and clinical heterogeneity within NCCN favorablerisk acute myeloid leukemia Leuk Res Yang JJ Park TS Wan TSK Recurrent cytogenetic abnormalities in acute myeloid leukemia Methods Mol Biol Coenen EA Zwaan CM Reinhardt D et al Pediatric acute myeloid leukemia with t816p11p13 a distinct clinical and biological entity a collaborative study by the InternationalBerlinFrankfurtMunster AMLstudy group Blood Xie W Hu S Xu J Chen Z Medeiros LJ Tang G Acute myeloid leukemia with t816p112p133KAT6ACREBBP in adults Ann Hematol Haferlach T Kohlmann A Klein HU et al AML with translocation t816p11p13 demonstrates unique cytomorphological cytogenetic molecular and prognostic features Leukemia Gervais C Murati A Helias C et al Acute myeloid leukaemia with 8p11 MYST3 rearrangement An integrated cytologic cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique Leukemia Diab A Zickl L AbdelWahab O et al Acute myeloid leukemia with translocation t816 presents with features which mimic acute promyelocytic leukemia and is associated with poor prognosis Leuk Res Schouten TJ Hustinx TW Scheres JM Holland R de Vaan GA Malignant histiocytosis Clinical and cytogenetic studies in a newborn and a child Cancer Brown T Swansbury J Taj MM Prognosis of patients with t816p11p13 acute myeloid leukemia Leuk Lymphoma Barrett R Morash B Roback D et al FISH identifies a KAT6ACREBBP fusion caused by a cryptic insertional t816 in a case of spontaneously remitting congenital acute myeloid leukemia with a normal karyotype Pediatr Blood Cancer Schumacher J Szankasi P Kelley TW Detection and quantification of acute myeloid leukemiaassociated fusion transcripts Methods Mol Biol DÃazBeyá M Navarro A Ferrer G et al Acute myeloid leukemia with translocation 816p11p13 and MYST3CREBBP rearrangement harbors a distinctive microRNA signature targeting RET protooncogene Leukemia Veigaard C Nørgaard JM Kjeldsen E Genomic profiling in high hyperdiploid acute myeloid leukemia a retrospective study of cases Cancer Genet Yasar D Karadogan I Alanoglu G et al Array comparative genomic hybridization analysis of adult acute leukemia patients Cancer Genet Cytogenet van der Veken LT Buijs A Array CGH in human leukemia from somatics to genetics Cytogenet Genome Res Laskowska J Szczepanek J StyczyÅski J Tretyn A Array comparative genomic hybridization in pediatric acute leukemias Pediatr Hematol Oncol Mehrotra M Luthra R Ravandi F et al Identification of clinically important chromosomal aberrations in acute myeloid leukemia by arraybased comparative genomic hybridization Leuk Lymphoma Kulikowski LD Bellucco FTS Nogueira SI et al Pure duplication 1q41qter further delineation of trisomy 1q syndromes Am J Med Genet A 2008146A2663 Nowaczyk MJM Bayani J Freeman V Watts J Squire J Xu J De novo 1q32q44 duplication and distal 1q trisomy syndrome Am J Med Genet A 2003120A229 Cone EB Dalton SS Van Noord M Tracy ET Rice HE Routh JC Biomarkers for wilms tumor a systematic review J Urol Xu B Ghossein R Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma Endocr Pathol Fournier A Florin A Lefebvre C Solly F Leroux D Callanan MB Genetics and epigenetics of 1q rearrangements in hematological malignancies Cytogenet Genome Res Lancman G Tremblay D Barley K et al The effect of novel therapies in highmolecularrisk multiple myeloma Clin Adv Hematol Oncol Bacher U Schnittger S Grüneisen A Haferlach T Kern W Haferlach C Inverted duplication dup1q32q21 as sole aberration in lymphoid and myeloid malignancies Cancer Genet Cytogenet Marcellino BK Hoffman R Tripodi J et al Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53 Blood Adv Beach DF Barnoski BL Aviv H et al Duplication of chromosome [dup1q21q32] as the sole cytogenetic abnormality in a patient previously treated for AML Cancer Genet Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | 2 |
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14 cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classiï¬ed into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in speciï¬c chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22 ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and ï¬broblastgrowth factor receptor FGFR gene fusions30 Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients beneï¬t from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classiï¬cation andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aï¬atoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver ï¬ukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difï¬culty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespeciï¬c survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Paciï¬c Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11 Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14 Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is aï¬rm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was ï¬rst described in cHCCICC was not systematicallydescribed until when it was classiï¬ed into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly deï¬ned areas type C mixedtype presents as intimate association without clear boundaries18 In another classiï¬cation system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identiï¬able intermediatetransition between HCC and ICC type III ï¬brolamellar tumors resemblesthe ï¬brolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classiï¬cationis commonly used in which cHCCICC is classiï¬ed into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a uniï¬ed classiï¬cation system greatly adds to the difï¬culty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdeï¬ned cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identiï¬ed stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA ampliï¬cations are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in of cases Recently some oncogenic genes were identiï¬ed in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identiï¬ed as a driver ofHCC45 VEGFA and CCND1FGF19 have also identiï¬ed in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identiï¬ed For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22 and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately of cases22 Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30 Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in of ICC cases32 Fusions ampliï¬cations translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC and ICC Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate ï¬lament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difï¬cult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166 Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efï¬cacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L ï¬rstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5ï¬uorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71 This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current ï¬rstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onï¬rstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand ï¬uoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the ï¬rstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe beneï¬t of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] conï¬dence intervalPFS months versus months p This effectiveness wasconï¬rmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the ï¬rstlinetreatment for advanced cholangiocarcinoma[CI] and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe ï¬rstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efï¬cacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efï¬cacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased beneï¬t for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095 Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI months CI months CI and months CI respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as ï¬rst line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir ï¬rstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the ï¬rst US Food and Drug Administration FDAapproved ï¬rstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPaciï¬c have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the ï¬rst generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have beneï¬tedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1 FGFR1 PDGFR RET and KIT In August theFDA approved lenvatinib for ï¬rstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the ï¬rstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modiï¬cations Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can speciï¬cally inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPaciï¬cREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further conï¬rmed the efï¬cacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival beneï¬t observed across all age subgroups and atolerable safety proï¬le supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety proï¬le apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic proï¬le and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a ï¬rstinhuman phase I study NCT02508467 of ï¬sogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123 Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identiï¬ed in recent years is the inhibitor of the ï¬broblast growth factorFGF signaling pathway which consists of members labeled FGF1FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1 FGF signals regulate cell proliferation in which FGFR2fusions occurred in of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the ï¬rst and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on ï¬ndings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may beneï¬t from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPï¬zeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial Identiï¬erTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi | 2 |
" child maltreatment leads to enormous adverse short and longterm health outcomes the aim ofthis study is to estimate the burden of disease and the cost of illness attributable to child maltreatment in japanmethods an incidencebased topdown cost of illness analysis was conducted to estimate medical costs andburden of disease attributable to child maltreatment based on a societal perspective the assessment includedshortterm and longterm medical costs and burden of disease measured by disabilityadjusted life years dalysthat generates mortality and morbidities based on several national surveys and systematic review we consideredthe main types of child maltreatment as exposure for which the incidence was obtained from literature reviewbased on population attributable fractions pafs burden of disease of physical and mental health consequencesattributable to child maltreatment were estimated then dalys were converted into monetary value the lifetimeeconomic burden was finally estimated by combining with medical costs and subject to sensitivity analysisresults the lifetime disease burden expressed in dalys was estimated at dalys ci dalys for the cohort victims in based on the incidence according to literature review the overall lifetimeeconomic burden was billion usd equivalent to million times of gross domestic product gdp per capitaamong the total economic burden costs of suffering and pain based on dalys were accounting for theseestimates were times of conservative estimates which used incidence data from official reported casess this study found that the national lifetime cost was huge and equivalent to million gdp percapita and its burden of disease was approximately equal to that of colon and rectum cancers or stomach cancerour findings particularly in terms of revealed the considerable burden of disease in long term and potential effectsof the strengthened maternal and child care as the preventive strategykeywords child maltreatment burden of disease study lifelong health consequences disabilityadjusted life yeardaly costofillness correspondence gairuoyanipssgojp1department of health policy national center for child health anddevelopment tokyo japan3department of empirical social security research national institute ofpopulation and social security research uchisaiwaicho chiyodakutokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmo bmc public health page of child maltreatment is a raising concern in public healthand social welfare in japan the reported number ofsuspected cases of child maltreatment is increasing from in to in according to theministry of health labour and welfare mhlw ofjapan child maltreatment is categorised into four essential types physical sexual or psychological includingwitnessing domestic violence wdv abuse and neglect exposure to multiple types and repeated episodes ofmaltreatment during childhood is associated with highrisks to enormous adverse health outcomes causing asignificant social and economic burden on individualsfamilies and societies those adverse outcomes duringchildhood include child death injuries and disabilitiesdevelopmental and behavioural problems moreover therelated physical and mental health conditions persistinto adulthood leading to the onset of chronic diseasesdepression drug alcohol misuse and risk sexual behaviour suicide ideation [ ]the number ofthe related analysis ofthe government has introduced a couple of protectivemeasures with increasing public budget [] assessment of costs and burden of disease helps developmentof resource allocation and priority setting in public sector paralleling with growing concerns on child maltreatmenttheprevalence health consequences and economic burdenis increasing so far for the economic burden there aretwo typical research frameworks one is a comprehensively costs evaluation from healthcare social educa[]tional areas and loss in productivity another one is to measure related economic and diseaseburden [ ] wada reported the socialcosts of child abuse in japan included direct costs ofdealing with abuse and the indirect costs related to longterm damage from abuse during the fiscal year onthe other hand the first framework is likely to underestimate longterm deleterious effects of child maltreatmenton which evidence derived from longitudinal studies isless available compared to that on the shortterm counterpart by integrating previous evidence our costofillness study aimed to assess lifetime economic anddisease burden of mortality and morbidities attributed tochild maltreatment based on the later frameworkinorder to address the evidence gap we extended cost calculations for monetary values converted from disabilityadjusted life years dalys covering related mortalityand morbidities methodsan incidencebased victims estimated by incidencetopdown approach or attributable risk approach measuring the proportion of a disease that is due to exposureto risk factor was applied in this study from a societalperspective we employed the following steps to estimate the total economic burden constituted by directand indirect costs population attributable fraction paf wasgenerated to estimate longterm impactscosts attributed to child maltreatment shortterm and longterm direct medical costs wereassessed by using national expenditure databasesindirect costs measured include productivity losscaused by abusive head trauma and economicburden deriving from dalys finally sensitivity analyses were performed for theplausible range of the discount rate and theincidence prevalenceestimating pafin the topdown approach paf for each disease i measured that how health outcomes and their associatedcosts may be attributed to child abuse using the following formula [ ]pafi ¼ p rri °p rri ¾¾ ¾ °p prevalence of child abuse rr the relative risk of theoutcome i in those who experienced child abuse compared with those who did notrisk ratio rr or odds ratio orseveral previous related systematic reviews and metaanalyses summarised the relevant health consequences[ ] as adverse childhood experiences acesoften intertwine with child maltreatment cluster in childrens lives and cumulatively lead to poor health outcomes we pooled the ors from a recent systematicreview and metaanalysis for the effect of multiple aceson health rather than that for each category of childmaltreatmentin japan thethe pooled prevalencea literature review was performed to synthesize the evidence on epidemiological characteristicsthe consequencesreview focused on thosepublished between december and march onmedline pubmed web of science scopus and ciniis japanese literature details of the search strategy search terms used and inclusion and exclusion criteria are provided in the additional file we combinedour review results with those studies in japan includedin an existing systematic review and calculated thesimple sizeweighted mean incidenceprevalenceinthe median value was also calculated toaddition 0cmo bmc public health page of examine the robustness supplementary table theannual incidence rate was obtained by the formula incidence rate ¼prevalenceaverage durationdue to the lack of local data on the average duration we adopted that published in australia theaverage years for physical abuse and years forsexual abuse based on this findingthe weightedaverage of years was used for other categories ofabusedirect medical costsshortterm medical costsfor abusive head trauma aht is the leading causeof death due to child abuse among children youngerthan years old we estimated its hospitalizationcosts as shortterm medical costs by multiplying theincidence of aht under years old the agespecific population in and admission medical fee per case there were two reported incidences one is the possibleincidence considering countable possibility ofaht cases at most and another one is the presumptiveincidence representing victims had intracranial injuriesor intentionalinjuries with certain icd10 code weused the possible incidence for the general calculationand the latter one in sensitivity analysis the total possible aht cases aged under years was about times ofthe presumptive counterparts longterm medical costsfor longterm medical costs we used national healthcare expenditures and patient survey tosimulate disease burden ofrelevant health consequences by sex and age group above and then multiplied with pafs to calculatethe attributable costs in the victim cohort of [ ] on the other hand we did not include selfharm and collective violence because of the limitationto distinguish the two in the reported overallinjurycasesindirect costsin this study we considered differential and loss of earning as a result of human capital depreciation is causedby mortality and morbidities it was presented as a monetary value of dalys and gdp per capita [ ]dalys and its monetary valuethe disease burden indicator daly aggregates yearsof life lost for premature death and years lost due todisability for morbidities related data wereobtained from the who global burden of diseasegbd using the pooled ors as described bykaren we matched each relatedhealth outcome with the cause of disease burdenin the who gbd categories though it was difficultto match some outcomes with the cause of gbdsupplementary table then monetary value was converted from daly attributable to child maltreatment by multiplying dalyand gdp per capita with adjustment of purchasingpower parity in productivity losses due to aht fatal casesproductivity loss due to fatal cases of child maltreatment was calculated based on the reported fatal caseswhich figure was obtained from official data andthe average lifetime income subject to discounting in there were abuserelated deaths reported injapan not including family suicide with the averageonset age of years the discounted lifetime income from to years old was calculated by assuming the longterm growth in labour productivityto be per year dalys losses of survival ahtfor disease burden due to survival aht we considered sequelae such as vision loss brain damage andreduced life span and longterm health consequences as developing diseases in adulthood we calculated the disease burden of aht in bymultiplying average cases and the estimated meanlifetime daly loss per case at different severity mildmoderate and severe longterm dalys losses of other diseasesthen the longterm health consequences were calculatedusing the following formuladaly losses ¼ Ï pafi 03original dalyi°i different child abuse related health outcome°¾¾sensitivity analysesa discount rate of is generally performed which wasrecommended in the domestic guideline for costeffectiveness analysis whereas especially in the usa discount rate of has been selected and applied inthe cost estimate reports of centers for disease controland a best practices for the social return on investmentanalysis recommended by experts and guidelines assuch the parameter potentially affects the finally resultswe adopted a plausible range of to for sensitivityanalysis 0cmo bmc public health page of in addition we also calculated costs and diseaseburden using the incidenceprevalence data based onofficially reported child abuse cases to calculate theconservative incidence of child abuse by categorieswe obtained the official data of victim cases reportedby child consultation facilities in and thendivided them by the total population number in corresponding age data by sex were not availablecocurrentinformation was not available and theoverlapped cases were not considered supplementarytable the initial victim age is assumed to be years old according to an ageweighted incidence calculation based on official reported cases we assumed the probable abuserelated death cases to be times of the reported cases based on the ratio of thepresumptive and the possible incidence of aht casesamong children aged under years resultsthe main results showed in tables were discounted at and conservative estimates were given for sensitivityanalysesthe pooled incidence prevalence and disease burdenthe estimations on different types of child maltreatment incidence draw from literature reviews variedregarding differences between sex except physicalabuse girls suffered more than boys in sexual abuseand witnessing domestic violence table the estimated lifetime disease burden associated with childmaltreatment onset in was considerable dalys with a ci of dalys to dalys table the top causes of totaldisease burden due to child abuse were suicide attempts cardiovascular disease and depression cancercostofillness analysis for child maltreatmenttable demonstrates lifetime costs attributed to childabuse onset in the total direct cost was estimatedtable estimated incidenceprevalence of child abuse in japanestimates aincidence bmalefemalephysical abuse sexual abuse psychological abusewdv c other d prevalencemalefemaleneglect a sample sized weighted mean valueb incidence rate prevalence average durationc wdv witnessing domestic violenced not specified as wdv often expressed as emotionalpsychological abuseto be usd million 95ci million11 million while the total indirect cost was estimatedto be usd million 95ci million52 million accounting for of the total lifetimecosts which were almost million times gdp percapita economic loss initiated from dalys in longterm costs of suffering and pain accounted for ofthe overall estimatessensitivity analysesconservative estimates based on the reported cases incidence showed a tendency similar to that observed in thedisease burden based on the literature review amongwhich psychological abuse including wdv accountedfor the majority of reported child abuse cases however the incidence estimated from the review weremuch higher than those reported by child protectionagencies the conservative estimation leading to about times difference gap on child maltreat burden bydifference discount rate table discussionour results indicated that disease and economic burdenattributable to child maltreatment is substantial in particular that originated from the longterm health consequences accounts for the majoritybased on literature review the pooled incidence ofchild maltreatment in japan is much higher than officially reported which is consistent with the findingsof other studies [ ] because of difficulty toidentify the actual cases and a public attitude to consider child abuse as a private affair in the society theofficially reported cases are likely to represent the tipof an icebergthe fourpsychological abuse including wdv representedthe majority of reported cases the results of the literature review also showed a gender difference in theprevalence oftypes of child abuse sizeweighted mean values girls were found to be morelikely to experience the harmful practices comparedto boys particularly sexual abuse this tendency wasalso observed in other countries in east asia and pacific region comparing those living in othercountries in the east asia and pacific region [ ]japanese children tended to less likely to experiencephysical abuse boys vs girls vsalthough it is difficult to directly compare the results across different study settings due to the different methodologies parameters and target populationsadopted the ingredients of the lifetime economic anddisease burden considered in our studyincludingmedical costs and monetary value of disease burdenare similar to that adopted in previous studies [ 0cmo bmc public health page of table longterm daly lost attributable to child abuse in japandiseases attributed to child abuse asuicide attemptdalys confidence intervalcancercardiovascular diseasedepressionrespiratory diseaseliver or digestive diseaseanxietyproblematic drug useabusive head traumaproblematic alcohol usediabetessexually transmitted infectionsviolence victimisationviolence perpetrationtotaldalys monetary value billion usa simple size weighted mean prevalence at discounted rate] still our results showed that the disease burdenwas about times of the conservative estimationdue to the huge gap of incidence generated from literature and that officially reported the number isconsistent with an australian research that showed awide distribution ofthe annual prevalence rangingfrom to in the conservative lifetimecourse simulation the initial victim age is assumed tobe years old according to an ageweighted incidencecalculation based on official reported cases whichwas also consistent with previous studies our study in particular highlighted dalys in longterm attributable to child maltreatment accountingin the overallfor a relevant proportion lifetime costs the estimation of disease burden attributed to child maltreatment dalys wascomparable to the total dalys due to colon and rectum cancers dalys in or stomachcancer dalys in to our knowledge this is the first study to estimatelifetime economic burden of child maltreatmentinjapan based on an epidemiological model the idea ofthis method is to convert diseaseinduced losses ofwellbeing into economic terms by multiplying theannual number oflost life years due to disease bysubreginal per capita income so far few studies hadever taken this part of costs into account potentiallyleading to an underestimation of health and economictable lifetime costs attributable to child abuse for the first time in ciitems of the costs usd milliondirect costs medical costsshortterm ahtlongterm other diseasesindirect costsabuse death a productivity lossessurvival aht dalys blongterm loss of other diseases btotal costsaht abusive head traumaa we used times of base line data for range costs of child abuseb costs of suffering and pain dalys converted into monetary value by multiplying a gross domestic product per capita million gdp per capita 0cmo bmc public health page of table sensitivity analyses on incidence resource and discounted ratesensitivity analysisliterature based estimation adisease burden in dalys 95cieconomic burden usd million 95cidr dr dr conservative estimation bdr dr dr dr discounted ratea estimated based on literature review simple size weighted average prevalenceb estimated based on the number of consultation cases disposed about child abuse at child guidance centres probable estimate of abuse death was assumedabout times confirmed aht casespossible cases of the costs of conservative estimatechild maltreatmentimpacts ofin addition weadopted conservative calculation methodology in thesensitivity analyses to estimate the burden of childmaltreatment for more reliable range estimationsthere are several limitations to this study first thecooccurrence of multiple types of child abuse isprevalent resulting in difficulties to identify theadverse effects separately in order to minimize possible consequent overestimation we used the pooledors of multiple adverse childhood health experiencesinstead of each types of child maltreatment and itsseverity second we focused on the economic burdendue to the mortality and morbidity of child maltreatment but did not consider nonhealth human capitalaspectslikeother economic burden estimation studies the availability of data on the related medical costs were limited wehealthconsequences and explored their unit costs for the estimates to address the knowledge gap thirdtargeted majorneverthelessthereproductiverecently in japan a continuum ofintensive supports to mothers and childrearing families encompassingcycle has been widelyimplemented in most local authorities such an integral approach serves as an essential preventive strategy against child maltreatment and other harmfulpractices by early detection and intervention of highrisk households in pregnancy postpartum and childrearing periods thisstudy can provide decisionmakers information on the economic burden of childmaltreatment as well as an important input in futureeconomic evaluations costeffectiveness analysis oncurrently ongoing intervention and policy in additionour results hint an emphasis on preventive interventions on suicide attempts and depression which aretop causes of the attributable disease burden due tochild maltreatmentour study demonstrated that lifetime disease and economic burden due to child maltreatment in japan is substantial its disease burden was approximately equal tothe burden of colon and rectum cancers or stomach cancer in particular it is important to include the longterm disease burden in future studies related to diseaseburden and cost of illness for both technical and policyperspectivessupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12889020093978additional file table a1 studies included in the quantitativesynthesis table a2 health outcomes and pooled ors used in this studyaht not included table a3 incidence rate by age and average onsetage based on the number of consultation cases disposed about childabuse at child guidance centersadditional file systematics review 2018520findpossible literature including japanese studies on risk of health outcomesattributable to child maltreatment figure a1 study selection prismaflow diagramabbreviationsdalys disabilityadjusted life years pafs population attributable fractionsgdp gross domestic product mhlw ministry of health labour and welfarewdv witnessing domestic violence aht abusive head traumaicd international classification of diseases gbd global burden of diseaserr risk ratio or odds ratio aces adverse childhood experiencesacknowledgementswe are grateful thank members of health informatics department kyotouniversity of public health school for their kind supportauthors contributionsmx and gr designed the study mx did the calculation and draft themanuscript gr and ty takahashi contributed to the revise ty tachibanatb and nt critically reviewed and provided important intellectual feedbackon the revise all authors have read and approved the manuscriptfundingthis study is granted by health labour sciences research grant japanagency for medical research and development and as part of an ipss 0cmo bmc public health page of project on the realization of japans plan for dynamic engagement of allcitizens the funders did not have any role in the study design datacollection and analysis interpretation of data or in writing the manuscriptavailability of data and materialsall the raw data is publicly accessible from respective official website asreference national healthcare expenditures and patient survey the datasets analysed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare no conflict of interestauthor details1department of health policy national center for child health anddevelopment tokyo japan 2department of health informatics kyotouniversity school of public health kyoto japan 3department of empiricalsocial security research national institute of population and social securityresearch uchisaiwaicho chiyodaku tokyo japan4maternalchild psychiatry department of psychosocial medicine nationalcenter for child health and development tokyo japan 5faculty ofeconomics saitama university sakuraku japanreceived march accepted august referencesgilbert r widom cs browne k fergusson d webb e sjtl j burden andconsequences of child maltreatment in highincome countries lancetnumber of consultation cases disposed about child abuse at child guidancecenters in japan in japanese [httpswwwestatgojpstatsearchfilespage1layoutdatalisttstat000001034573cycle8tclass1000001108815tclass2000001108820second21]definition and present condition of child abuse in japanese [httpswwwmhlwgojpseisakunitsuitebunyakodomokodomo_kosodatedvabouthtml] accessed july currie j spatz widom c longterm consequences of child abuse andneglect on adult economic wellbeing child maltreatment hughes k bellis ma hardcastle ka sethi d butchart a mikton c jones ldunne mp the effect of multiple adverse childhood experiences on healtha systematic review and metaanalysis lancet public health 201728e356fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai11901000koyoukintoujidoukateikyokusoumuka002_1pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile06seisakujouhou11900000koyoukintoujidoukateikyoku0000180499pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai12601000seisakutoukatsukansanjikanshitsu_shakaihoshoutantou0000058633pdf] accessed july the economics of child abuse a study of san francisco [httpssafeandsoundwpcontentuploads201709economicsofabuse_report_sfcapc1pdf] accessed july fang x brown ds florence cs mercy ja the economic burden of childmaltreatment in the united states and implications for prevention childabuse negl habetha s bleich s weidenhammer j fegert jm a prevalencebasedapproach to societal costs occurring in consequence of child abuse andneglect child adolesc psychiatry ment health mccarthy mm taylor p norman re pezzullo l tucci j goddard c thelifetime economic and social costs of child maltreatment in australia childyouth serv rev wada i igarashi a the social costs of child abuse in japan child youth servrev miller tr steinbeigle r wicks a lawrence ba barr m barr rgjp disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e1545fang x fry da brown ds mercy ja dunne mp butchart ar corso psmaynzyuk k dzhygyr y chen y the burden of child maltreatment inthe east asia and pacific region child abuse negl corso ps fertig ar the economic impact of child maltreatment in theunited states are the estimates credible child abuse negl macroeconomics and health investing in health for economicdevelopment [httpwhqlibdocwhointpublications2001924154550xpdf]accessed july segel je costofillness studiesa primer rtiunc center of excellence inhealth promotion economics jo c costofillness studies concepts scopes and methods clin molhepatol metrics population attributable fraction paf [httpwwwwhointhealthinfoglobal_burden_diseasemetrics_pafen] accessed july norman re byambaa m de r butchart a scott j vos t the longtermhealth consequences of child physical abuse emotional abuse and neglecta systematic review and metaanalysis plos med 2012911e1001349kalmakis ka chandler ge health consequences of adverse childhoodexperiences a systematic review j am assoc nurse pract unicef child maltreatment prevalence incidence and consequences inthe east asia and pacific region new york unicef rothman kj epidemiology an introduction oxford university press joyce t huecker mr pediatric abusive head trauma shaken babysyndrome [updated aug ] in statpearls [internet] treasure islandfl statpearls publishing available from httpswwwncbinlmnihgovbooksnbk499836 yamaoka y fujiwara t fujino y matsuda s fushimi k incidence and agedistribution of hospitalized presumptive and possible abusive head traumaof children under months old in japan 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devices injapan value health moore se scott jg ferrari aj mills r dunne mp erskine he devries kmdegenhardt l vos t whiteford ha burden attributable to childmaltreatment in australia child abuse negl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
" medical practice variation in caesarean section rates is the most studied type of practice variation inthe field of obstetrics and gynaecology this has not resulted in increased homogeneity of treatment betweengeographic areas or healthcare providers our study aim was to evaluate whether current study designs on medicalpractice variation of caesarean section rates were optimized to identify the unwarranted share of practice variationand could contribute to the reduction of unwarranted practice variation by meeting criteria for audit and feedbackmethods we searched pubmed embase ebscocinahl and wileycochrane library from inception to march24th studies that compared the rate of caesarean sections between individuals institutions or geographicareas were included study design was assessed on selection procedure of study population data source casemixcorrection patient preference aggregation level of analysis maternal and neonatal outcome and determinantsprofessional and anizational characteristicsresults a total of studies were included most studies measured the caesarean section rate in the entirestudy population instead of using a sample national databases were most often used as information source casemix correction was performed in studies the robson classification was used in of thestudies following its endorsement by the who in the most common levels of aggregation were hospitallevel and grouped hospitals eg private versus public the percentage of studies that assessed therelationship between variation in caesarean section rates and maternal outcome was neonatal outcome determinants professional and anizational characteristics and patient preference s study designs of practice variation in caesarean sections varied considerably raising questions abouttheir appropriateness studies focused on measuring practice variation rather than contributing to the reduction ofunwarranted practice variation future studies should correct for differences in patient characteristics casemix andpatient preference to identify unwarranted practice variation practice variation studies could be used for audit andfeedback if results are presented at lower levels of aggregation and appeal to intrinsic motivation of physicians forexample by including the health effects on mother and childkeywords caesarean section medical practice variation study design characteristics correspondence mdhvinkvunl mdhvinkgmailcom1department health sciences faculty of science talma institute vrijeuniversiteit de boelelaan hv amsterdam the netherlands2department of obstetrics and gynaecology university medical centergroningen groningen the netherlandsfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cvink bmc pregnancy and childbirth page of the caesarean section has been the most performed surgical procedure worldwide for many decades it hasbeen extensively studied both to optimize treatment and to understand why deviations from optimal treatment occur this longterm popularity has not resulted in evidence based homogeneoustreatmentbetween geographic areas or healthcare providers [ ]the magnitude of the variation has raised questionsabout regional differences in quality of healthcare especially in countries with similar resources moreover years of study on practice variation shows no trend ofincreasing regional l one worldwide conformity variation in medical practice can be divided in warranted and unwarranted practice variation variationis warranted when it results from variation in need forexample due to varying rates of extreme premature deliveries extremely preterm deliveries are centralized atinstitutions with the highest expertise of neonatal careas it yields the most optimal outcome these institutions may deviate from the national average as theyserve a highrisk populationmedical practice variation is unwarranted if it cannotbe explained by patient characteristics or patient preference [ ] to identify unwarranted practice variationstudies should compare study groups that are comparable in terms of relevant patient characteristics or makethem comparable through careful casemix correction patient preference is important when both modesof delivery vaginal delivery and caesarean section arean acceptable option variation of caesarean sectionrates is desirable to allow for differences in patient preference across healthcare providers and random or unmeasured differences in need of having a caesareansection when a reported rate deviates more from an acceptable range differences may less likely be attributableto differences in patient preference as both over andundertreatment of caesarean sections harms mother andchild it is therefore likely that quality of healthcare formother and child can be improved by reducing unwarranted practice variation of caesarean sections sufficientevidence on risks and benefits of caesarean sections mayhelp to reduce variation higher quality evidence will result in better guidance on the optimal caesarean sectionrate for specific obstetric conditions subsequentlyuptodate clinical guidelines and clinical support systems may facilitate clinicians to implement recent evidence finally shared decision making should beincorporated in daily clinical practice to empower patients to decide what suits them best improving the process of generating evidence implementing clinical guidelines and incorporating shareddecision making requires healthcare professionals tochange their clinical behavior which is complex auditand feedback is a nonclinical intervention that supportschange of clinical behavior literature shows thathealthcare professionals may be encouraged with information relevant to them one example is performancefeedback on a low level of aggregation ie group or individual level another is to tap into the intrinsic motivation to do well for patients evidence shows thatunnecessary caesarean sections cause an increase in maternal death rates and may affect infant health negatively monitoring and reporting mother and child healthmay motivate change to reduce unnecessary caesareansections audit and feedback has been put forwardas a way through which research can contribute to thereduction of practice variation but it is unclearwhether current research designs of studies on medicalpractice variation of caesarean section rates can be usedfor this purposemedical practice variation in caesarean section rateshas been extensively studied studies started as earlyas and the interest for the topic has remainedstrong however unwarranted medical practicevariation of caesarean section rates has not been reduced the objective of this scoping review is to evaluatewhether studies on medical practice variation of caesarean section rates have used an optimal study design toidentify unwarranted practice variation and when identified can also be used for audit and feedback to contributereduction of unwarranted practicevariationto themethodsto evaluate the characteristics of all caesarean sectionvariation studies we opted for a scoping review wefollowed the prisma statement the researchprotocol was not publishedsearch strategywe searched studies that compared caesarean sectionrates between individual healthcare professionals hospitals groups of hospitals or geographic areas a comprehensive search was performed in collaboration with amedical librarian in the bibliographic databases pubmedembase ebscocinahl and wileycochrane libraryfrom inception up to march 24th the followingterms were used including synonyms and closely relatedwords as index terms or freetext words practice patterns caesarean section the freetext term caesarean section was only used in titles the meshtermpractice patterns includes several descriptions of practice variation the search strategy was performed without date orsearchlanguage restriction the full 0cvink bmc pregnancy and childbirth page of strategies for all databases can be found in additional filestudy selectionall studies that reported on any variation in caesareansection rates between individual healthcare professionals hospitals groups of hospitals or geographicareas were included we included any type of study designie crosssectional study designs and both prospective and retrospective longitudinal studiesexclusion criteriawe excluded studies that were not published in englishand studies that did not publish data on variation of caesarean section rates between healthcare professionalshospitals groups of hospitals or geographic areasprocess of study identification and selectiontitles and abstracts were downloaded and entered inendnote version x81 duplicates were removed tworesearchers mv and pdb screened titles and abstractsthe researchers independently decided whether to include the for full text screening disagreementwas resolved by consensus if no consensus could beachieved a third researcher made the decision evdhfull text screening was performed by two researcherswho decided independently whether to include the or not mv and pdb in case of disagreement thethird researcher evdh decided whether the metthe predefined inclusion criteria data on the designcharacteristics of the studies were extracted by one researcher mv these data were randomly crosscheckedby a second researcher pdbdata extractionto describe the variation of caesarean section rates wescored the minimum and maximum caesarean sectionrate when caesarean section rates of multiple yearswere reported the rate of the most recent year was usedas an indicator for the risk of selection bias we scoredthe selection of study population we differentiated between the use of a study sample or the entire studypopulation to estimate the caesarean section rate theuse of a study sample was considered as a high risk ofselection bias if the study lacked a description of thesampling frame the assessment of the caesarean sectionrate of the entire study population was considered as alow risk of selection bias to indicate the risk of information bias we differentiated between the use of electronic patientepf a national database orquestionnaires the use of epf and databases were considered as low risk of bias as the information was collected by attending healthcare professionalsfilesidentification of unwarranted practice variationto identify whether studies distinguished between warranted and unwarranted practice variation we scoredwhether casemix correction was performed and if patient preference was taken into accountno restriction was imposed on the method of casemix correction examples of casemix corrections include calculating an adjusted or expected caesarean section rate reporting stratified odds ratios by patientcharacteristics or using logistic regression to adjust forpatient characteristics we extracted which variableswere used for casemix correction and whether the robson classification was used the latter is the system proposed by the world health anisation who andthe international federation of obstetrics and gynaecology figo to classify caesarean section casemix no restriction was imposed on how patient preferencewas measured measuring patient preference requiresadditional data collection this could be unfeasible forlarge cohort studies unless a truly random sample of sufficient size is used we assessed whether all studies tookpatient preference into account registered the cohortsize and noted how patient preference was measured ifpatient preference was measured we assessed whether asample was used and whether it was randomusefulness for audit and feedbackto evaluate whether the studies were able to providehealthcare professionals feedback on their clinical behavior in order to reduce unwarranted practice variationwe extracted the aggregation level of analysis that wasused and differentiated betweenindividual physiciangroup of physicians hospital hospital category regionor country similarly we scored whether maternal andneonatal outcomes were measured as outcome reporting informs healthcare professionals on their clinicalperformance we extracted all reported variablesin addition we extracted several explanatory factorsthat might contribute to unwarranted practice variationincluding anizational characteristics ie profitstatusor teachingstatus of the hospital and physician characteristics ie physician gender and age furthermore wescored whether studies analysed financial consequencesof unwarranted practice variation of caesarean sectionratesresultsthe process of study identification and selection is schematically presented according to the prisma statementin fig a total of records were identified from pubmed from embase from cochraneand from cinahl after duplication we screened abstracts for eligibility in total s were 0cvink bmc pregnancy and childbirth page of fig prisma flowchartfig average of the reported minimum and maximum caesarean section rate per year is not presented in fig as only studies areonly included until 24th of march 0cvink bmc pregnancy and childbirth page of selected for fulltext screening we excluded studiesthe reasons for exclusion are presented in fig intotal studies met the inclusion criteria and were included the included studies and their design characteristics and reported variation in caesarean section ratesare listed in additional file the included studies were published between and the cohorts that were studied varied between and more than million women most studies analyzed variation of caesarean section rates in the unitedstates studies followed by brazil studies andaustralia studies the number of studies per country are shown in additional file a wide variation incaesarean section rates is reported some subsaharanregions perform caesarean sections in less than ofthe deliveries by contrast the reported caesarean section rate of some municipalities in brazil reached the variation of caesarean section rates did not decrease over time figure shows the average reportedminimum and maximum caesarean section rates peryear the outlier in is one study that reported variation between four hospitals in rio de janeiro in oneprivate hospital more than of the women deliveredby a caesarean section in a similar situation occurred only two studies were included both reportingon variation in indiain studies a sample of the study populationwas used to estimate the caesarean section rate the majority of the studies studies measured themode of delivery of the entire study population bothmethods were used in eight studies and the selection frame was unclear in nine studies an exampleof a study in which both methods were used was theanalysis of variation in caesarean section rates betweencountries some country estimates were based on a sample of their population while others were based on registries ofin the samplebasedstudies studies defined a selection frame designed to select a representative samplethe entire populationthe majority of studies used data from registries suchas national databases studies or electronicpatient files studies questionnaires were usedin studies eg the demographic and healthsurvey dhs that was used in studies suchquestionnaires were sent to a sample of households inorder to collect information on live births of the pastyears in studies multiple data sources wereused or the data source was not describedidentification of unwarranted practice variationcasemix correction was performed in studies the variables that were used for casemix correction areshown in additional file baseline patient characteristics were observed in studies some studies didnot describe patient characteristics per cohort but didperform a correction for maternal or neonatal characteristics many different maternal and obstetric variables were used as baseline characteristic or for casemix correction age parity gestational age birthweight and maternal education level are the characteristics that were used most often morethan half of the variables were only used in one or twostudiesto reduce this heterogeneity and to increase the quality of casemix correction the who in recommended to use the robson classification as the standardfor casemix correction for studies on caesarean sectionrates in the period out of studiesused the robson classification four of these studies did perform additional casemix correction byusing specific patient characteristics the who notesthat the robson classification should especially be usedwhen comparing caesarean section rates between healthcare facilities or within healthcare facilities over time in the period studies compared caesarean section rates between healthcare providers individuallevel andhospital category of which used the robson classification within the same period of the studiesthat compared caesarean section rates between geographic areas used the robson classification thepercentage of studies that corrected for casemix did notchange over time figure shows the number of studiesthat corrected for casemix by yearlevel group of physicians hospitalthe effect of casemix correction is shown in fig of the studies that corrected for casemix studies calculated an adjusted caesarean sectionrate figure shows these rates per study categorizedper aggregation level the remaining studies calculated an expected caesarean section rate reportedstratified odds ratios by patient characteristics or usedlogistic regression to adjust for patient characteristicsat provider level individual physician group of physicians and hospital level of the studies and at geographic level regional or national of the studiescorrected for casemix figure shows that at the provider level casemix correction had a substantial impacton the provider caesarean section rate at the geographic level the impact of casemix correction wascomparatively smallsix studies took patient preference into accountthese studies did not assess variation of caesarean section rates for a specific obstetric condition for instancepatients with a history of caesarean section in which acaesarean section and vaginal delivery are both an acceptable option all studies measured patientpreference by questionnairesthat were handed tomothers that gave birth between one day and years 0cvink bmc pregnancy and childbirth page of fig number of studies per year with and without casemix correction is not presented in fig as only studies are only included until24th of march rates was least often studied at the level of the individualphysician and group of physicians no clear timetrend was observed in the choice of aggregation level ortrend in observed variation based on the level of aggregation the largest variation in caesarean section rates is reported on both the lowest ie individual level and thehighest ie international level of aggregationneonatal outcomes were captured in andmaternal outcomes in of the studies all variables that were used to measure these outcomes arelisted in additional file many different variables wereused to measure neonatal and maternal outcomes neonatal mortality apgar score maternal mortalityandhaemorrhage are outcomes that were measured mostoften half of the outcome variables were used in justone single study table shows the numbers of studiesper aggregation level that took neonatal and maternaloutcome into account neonatal and maternal outcomeswere most often reported if variation of caesarean nicu admissionprior to the survey patient preference was assessed byposing a variety of questions on the reason of the caesarean section and perceived influence on decision makingno studies reported the quality of decision making in of the studies a sample of the study populationwas used to measure patient preference samples variedbetween and women and three studies used apredefined sampling frameusefulness for audit and feedbacktable shows the study characteristics by aggregation levelthe majority of the studies used one aggregationlevel a minority used two and only three studies used three aggregation levels healthcare providersindividual physicians group of physicians hospitals or hospital categories were compared in studies andgeographic areas national or regional in studies hospitals and grouped hospitals eg private vspublic hospitals were most often used as aggregation levelof analysis medical practice variation of caesarean sectionfig the effect of casemix correction on different aggregation levels 0cvink bmc pregnancy and childbirth page of table study characteristics by aggregation levelnumber of studiesaverage cohort sizemedian cohort sizeentire population measured instead of samplereported variation in caesarean section rate average ofstudiesreported variation in caesarean section rate median ofstudiescasemix correctionmedical outcomeindividuallevelgroup ofphysicians hospitallevelhospitalcategoryregionnational sections was studied atphysiciansthe level ofthe individualdeterminants and financial consequencesa limited number of studies explored determinants toexplain medical practice variation of caesarean sectionrates in an additional analysis hospital characteristics orphysician characteristics were used in studies to explain differences in caesarean section rates thevariables that were used are listed in additional file financial consequences of unwarranted variation of caesarean section rates were calculated in six studiesdiscussionalmost four decades have past and studies werepublished following opits first report on variation ofcaesarean section rates between geographic areas inaustralia clearly the issue raised by the first studies hasnot lost its sense of urgency among researchers nor forthe funders of research because the magnitude of unwarranted variation was considered problematic andremained stable over time while previous reviewsnarrowed their focus on measuring variation betweengeographic areas [ ] or studied the difference between public and private hospitals the focus of thisreview was on the presence of study characteristics thatmay help to reduce unwarranted variation of caesareansection ratesstrengths and limitationsa strength of our review was the systematic search andselection procedure which allowed us to identify almost all studies on medical practice variation that compared caesarean section rates this resulted in a largenumber of included studies a second strength is thehigh level of detail of our analysis the selection of theindividual variables that were used for casemix correction outcomes or determinants enabled us to present anindepth overview of study characteristicsseverallimitations should be addressed first weaimed to describe study characteristics of all relevantpublished studies ie irrespective of the quality in theenglish language and therefore did not perform a qualityassessment of the included studies second we were unable to retrieve all publications that were selected forfulltext screening in order to limit the number of missing s we contacted the authors of missing sby email or through researchgate however this wasnot successful for of the studiesinterpretationfirst we appraised whether studies were designed to distinguish between warranted and unwarranted practicevariation by performing casemix correction and analysing patient preference casemix correction is an essential aspect of the quality of studies on practice variationbecause without casemix correction it remains unclearwhat share of the variation is attributable to health differences between populations and thus to what extentthe variation is warranted our results show that only of the studies that compared caesarean section ratesbetween healthcare providers performed casemix correction casemix correction was performed by calculating adjusted rates expected rates stratified odds ratiosor logistic regression analysis over time we observed noimprovement in the performance of casemix correctionpatient preference another important aspect to identify unwarranted practice variation was only measured insix studies without the assessment of patient preference it remains unclear whether practice variation canbe explained by differences in the outcome of shared decision making this is especially important when bothmodes of delivery vaginal delivery and caesarean section are an acceptable option patients with a historyof caesarean section breech or twin delivery are examples in which information on patient preference is necessary to differentiate between warranted and unwarrantedmedical practice variation [] our results show 0cvink bmc pregnancy and childbirth page of that none of studies that assessed variation in these specific obstetric situations patient preference was analysedto improve the identification of unwarranted practicevariation future studies should not only measure patientpreference but should focus on the implementation ofshared decision making recent literature shows thatseveral shared decision making measures are availablewhich could be included in the study design of medicalpractice variation studies to improve comparability both within healthcare facilities and between them robson proposed what latercame to be known as the robson classification systemfor assessing monitoring and comparing caesarean section rates in the who and the figo jointly endorsed this classification as the international standardfor casemix correction [ ] our data show that following the publication of this guideline by the who in the robson classification was only used in of all studies comparing providers and in studies comparing regions literature shows that casemixcorrection can be improved even more if additional patient characteristics are considered only of thestudies that were published between and andused the robson classification did perform additionalcasemix correction ie age adjusted caesarean sectionrates within robson groups studies that performedcasemix correction with or without robson classification used a wide variation of maternal and obstetriccharacteristics to identify unwarranted practice variation we advise to at least use the robson classificationand to standardise variables for additional casemix correction a delphi procedure can help obstetricians andmidwives to reach consensus on which variables to use the necessity to which casemix correction is neededdepends on the level of aggregation the lower the levelof aggregation the more case mix correction contributesto a valid description of clinical performance health care providers often operate in a network andtreat a subset of the entire population that subset ismore likely to differ between providers as they get morespecialized and the casemix differences may justify differences in caesarean section rates this requiresthat studies aimed at lower levels of aggregation placemore emphasis on casemix correction reporting standards and appropriate small number statistics once casemix has been appropriately controlled forfuture studies should aim to decompose regional unwarranted variation into lower levels of aggregation thisdecomposition allows regional variation to be attributedto health care providers however disaggregation ofcontributions to lower levels of aggregation is not without major risk on its own as groups of physicians getsmaller their clientbase may diverge more from theregional averagefor example due to specializationreporting differences or chance within providers eghospitals further disaggregation to the physician levelcould help identify individual contributions while thelower number of observations may harm both validityand reliability they may also provide a stronger stimulusfor change if the information is interpreted intelligentlyand presented in a motivating environmentstimulus for change might be further enhanced whenoutcome reporting becomes integrated in future studydesigns healthcare professionals are intrinsically motivated to deliver the best healthcare for their patients reporting outcomes in casemix corrected feedbackinformation directly stimulates the intrinsic motivationto improve outcome for patients if it becomes visiblethat increased caesarean section rates do not yield improved maternal and neonatal outcomes professionalsmight be encouraged to adapt clinical behavior our results show that the wide variation in outcome variablesdemands consensus and standardization studies will become more comparable and better interpretable when astandard set of outcomes is used for maternal infectiousmorbidity outcomes after caesarean delivery a core outcome set cos is developed we encourage to develop a cos for neonatal outcomes after caesareansectionforty years of research on caesarean section rates havebeen unable to reduce unwarranted practice variationour study shows that most studies do not meet the criteria to identify unwarranted practice variation and cannot be used for audit and feedback to contribute to thereduction of unwarranted practice variation future studies should correct for differences in patient characteristics and patient preference present results at low levelsof aggregation and appeal to intrinsic motivation by including the health effects on mother and childsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12884020031693additional file search strategies additional file contains the searchstrategies that were used for the databases pubmed embase ebscocinahl and wileycochrane libraryadditional file list of included studies additional file contains a listof all studies that were included in this scoping review per study theresults are summarized we used the following definitions for theindependent variables used in the table year year of publication author first author of study included title title of study included study period years from which caesarean section rates were reported inexample if a researcher performed a questionnaire survey in andincluded deliveries from y prior to the survey we reported study period caesarean section rate unadjusted caesarean section rate ofmost recent year reported cohort size the cohort size from which thecaesarean section rate is calculated if caesareans section rates from 0cvink bmc pregnancy and childbirth page of multiple years were reported we noted specifically the cohort size of thecohort that was used to calculate the most recent caesarean section rate data source data source that was used by the authors to calculate thereported caesarean section rate it is reported as other if data source isunknown or multiple data sources are used casemix correction thestudy reported an adjusted caesarean section rate expected caesareansection rate reported stratified odds ratios by patient characteristics orused logistic regression to adjust for patient characteristics yn aggregation level aggregation level of analysis outcome outcomes maternal or neonatal were noted yn determinants anisational orphysician characteristics were used to explain reported difference in caesarean section rates between healthcare professionals hospitals groupsof hospitals or geographic areas ynadditional file studies per country additional file describes thenumber of studies on medical practice variation of caesarean sectionrates that were conducted in each count | 0 |
"Two methods were used to inhibit the enzyme activity or protein expression of NQO1 an NQO1 inhibitor and NQO1 siRNA knockdown. Dicoumarol has been previously used to specifically inhibit the expression and activity of NQO1 [44]. As shown in pretreatment of cells with 100 or 250 µM sulindac (A) sulindac sulfone (B) or sulindac sulfide (C) followed by addition of 2 µM ?-lapachone for 12 h increased the cytotoxicity of ?-lapachone for both CL1-1 and CL1-5 cells and these effects were significantly reduced by addition of 10 µM dicoumarol. .0088122.g007 The increase in ?-lapachone-induced cell death caused by sulindac and its metabolites is blocked by the NQO1 inhibitor dicoumarol. CL1-1 cells (left) or CL1-5 cells (right) were left untreated or were pretreated for 6 h with 100 or 250 µM sulindac (A) sulindac sulfone (B) or sulindac sulfide (C) with or without 10 µM dicoumarol then were incubated for a further 12 h with or without addition of 2 µM ?-lapachone then cell survival was measured by crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p<0.05. Using siRNA knockdown of NQO1 at days 1 to 3 after NQO1 siRNA transfection of CL1-1 and CL1-5 cells no change in cell growth or cell morphology was noted (Figure S6). Efficiency of knockdown in CL1-1 and CL1-5 cells was demonstrated for RNA expression by RT-PCR (A) and realtime-PCR (Figure S7) and for protein expression by western blotting (B and C) showing that NQO1 siRNA significantly downregulated NQO1 expression. As shown in D NQO1 siRNA transfection significantly inhibited the increase in NQO1 enzyme activity induced in CL1-1 cells by incubation for 6 or 24 h with 100 or 250 µM sulindac (left panel) sulindac sulfone (center panel) or sulindac sulfide (right panel). When cells transfected for 24 h with siNQO1 or control siRNA were pretreated for 6 h with sulindac or its metabolites then cotreated for 12 h with drug plus 2 µM ?-lapachone the percentage cell survival results showed results that transfection with NQO1 siRNA caused a significant decrease in the cytotoxicity of combinations of ?-lapachone with sulindac (A) sulindac sulfone (B) or sulindac sulfide (C). These results showed that NQO1 plays an important role in the increase in ?-lapachone-induced cell death caused by sulindac or its metabolites. .0088122.g008 The knockdown effects of NQO1 siRNA on NQO1 RNA protein and activity. (AC) CL1-1 cells (left) or CL1-5 cells (right) were transfected for 1 to 3 days with control siRNA (CTL) or siRNA targeting NQO1 then RNA expression was measured by PCR (A) and protein expression by Western blotting (B and C). (D) CL1-1 cells transfected for 2 days with control siRNA or NQO1 siRNA were incubated alone or with 100 or 250 µM sulindac sulindac sulfide or sulindac sulfone for 6 or 24 h then NQO1 activity was measured. * : p<0.05 ***: p<0.001 compared to the identically treated cells transfected with control siRNA. .0088122.g009 NQO1 siRNA transfection significantly inhibits the effect of sulindac and its metabolites on ?-lapachone-induced cell death. CL1-1 cells (left) or CL1-5 cells (right) were transfected with control siRNA (?) or NQO1 siRNA (+) for 24 h then were left untreated or were incubated for 6 h with 100 or 250 µM sulindac (A) sulindac sulfone (B) or sulindac sulfide (C) then 2 µM ?-lapachone or medium was added and the cells incubated for 12 h when cell survival was measured using crystal violet staining and expressed as percentage survival compared to the untreated cells. * : p<0.05 for the indicated comparison. Discussion ?-lapachone Triggers Lung Cancer Cells to Undergo Apoptosis through an Increase in Intracellular Calcium Levels Increased JNK Activation Decreased Activation of PI3K ERK and AKT and a Decrease in the MMP Cell proliferation and cell death are under complex and precise control. Normally the proteins involved in cell proliferation survival or cell death are in a remarkable balance and unbalanced survival and apoptotic signals may lead to cell death. In most cells proliferation is mainly regulated through PI3K AKT and ERK [46] and cell death is regulated through another pathway involving JNK and p38 [47] [48]. JNK has recently been reported to be an important mediator in the ?-lapachone-induced cell death of breast and prostate cancer cells [45] [49]. ?-lapachone also triggers cell death of many cancer cells by increasing calcium signaling [6] [50]. Calcium the key messenger molecule in cells plays an important role in many signaling pathways and an imbalance in intracellular calcium levels causes abnormal cell function and leads to cell death. Treatment of cells with the intracellular calcium chelator BAPTA partially inhibited ?-lapachone-induced cell death showing that calcium was involved (). Activation of the cell death signal JNK (A) and inhibition of the cell survival signals p-PI3K p-AKT and p-ERK (A and B) were also observed in ?-lapachone-mediated lung cancer cell death showing that the MAP kinase signaling pathway is involved in the anticancer effect of ?-lapachone. Although ROS could have caused the cell death induced by ?-lapachone there was no change in intracellular H2O2 levels following ?-lapachone treatment (Figure S1D). However the MMP was dramatically decreased following ?-lapachone treatment (Figure S3C) suggesting that other ROS species might be involved in the ?-lapachone-induced cell death process. NQO1 is a Key Factor in the ?-lapachone-induced Lung Cancer Cell Death High NQO1 activity and expression are seen in many human tumors including carcinoma of the liver [51] [52] colon [53] breast [52] [54] brain [55] and lung [52] and NQO1 has been shown to be an important factor in ?-lapachone-induced cell death in many kinds of cancer cells [9] [44] including breast cancer [2] glioma [56] and prostate cancer [44]. In this study we demonstrated that the cytotoxicity of ?-lapachone for three different lung cancer cell lines was positively correlated with their NQO1 expression and enzyme activity (). Inhibition of NQO1 activity using the NQO1 inhibitor dicoumarol (Figure S3) blocked the ?-lapachone-induced increase in intracellular calcium levels (Figure S3) increase in p-JNK levels (A) and decrease in p-ERK p-PI3K and p-AKT levels (A and B). These results indicated that the balance between survival and death signals in lung cancer cells was disrupted by the decrease in p-PI3K p-AKT and p-ERK levels and the increase in p-JNK levels caused by ?-lapachone treatment and that NQO1 expression and activity were involved in the activation of all these apoptotic signals. Anti-inflammatory Drugs Increase NQO1 Levels and Enzyme Activity in Lung Cancer Cells Many drugs or treatments such as cisplatin [5] heat shock [19] or radiation [57] can increase NQO1 expression or activity and facilitate the cytotoxicity of ?-lapachone for various cancer cells. However such drugs or treatments are usually harmful to normal cells as well as cancer cells so there is a need for drugs or treatments that can facilitate the anti-cancer effect of ?-lapachone while being less harmful for normal cells. Sulindac has been shown to be a potent chemo-protective agent against colorectal cancer in both human and animal models [25] while sulindac sulfide [18] and sulindac and its two metabolites [18] [28]] have been reported to upregulate the expression of carcinogen detoxification enzymes including NQO1. It is known that sulindac compounds inhibit the activity of COX-1 and COX-2 and thus block the biosynthesis of prostaglandins [58]-[60]. In in vivo studies sulindac has been shown to be reversibly reduced to sulindac sulfide which can be irreversibly oxidized to sulindac sulfone all three of which are anti-inflammatory. Since 1995 several clinical trials have established that sulindac is effective at reducing the number and size of polyps in patients with familial adenomatous polyposis a precursor to colorectal cancer (NCI-P97-0110 NCI-P00-0150 [61])." | 1 |
" to improve the postoperative prognosis of patients with lung cancer predicting the recurrence highrisk patients is needed for the efficient application of adjuvant chemotherapy however predicting lung cancerrecurrence after a radical surgery is difficult even with conventional histopathological prognostic factors thereby anovel predictor should be identified as lipid metabolism alterations are known to contribute to cancer progressionwe hypothesized that lung adenocarcinomas with high recurrence risk contain candidate lipid predictors this studyaimed to identify candidate lipid predictors for the recurrence of lung adenocarcinoma after a radical surgerymethods frozen tissue samples of primary lung adenocarcinoma obtained from patients who underwent a radicalsurgery were retrospectively reviewed recurrent and nonrecurrent cases were assigned to recurrent n andnonrecurrent n groups respectively extracted lipids from frozen tissue samples were subjected to liquidchromatographytandem mass spectrometry analysis the average total lipid levels of the nonrecurrent andrecurrent groups were compared candidate predictors were screened by comparing the folding change and pvalue ofttest in each lipid species between the recurrent and nonrecurrent groupsresults the average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp a total of lipid species were increased folding change ¥ p and lipid species were decreasedfolding change p in the recurrent group among these candidates increased sphingomyelin smd351 inthe recurrent group was the most prominent candidate predictor showing high performance of recurrence predictionauc sensitivity specificity accuracy we propose smd351 as a novel candidate predictor for lung adenocarcinoma recurrence our finding cancontribute to precise recurrence prediction and qualified postoperative therapeutic strategy for lung adenocarcinomastrial registration this retrospective study was registered at the umin clinical trial registry umin000039202 on 21stjanuary keywords lung adenocarcinoma prognostic factor recurrence prediction lipid mass spectrometry correspondence kahyohamamedacjp1department of cellular and molecular anatomy hamamatsu universityschool of medicine handayama higashi ward hamamatsu shizuoka japan5international mass imaging center hamamatsu university school ofmedicine handayama higashi ward hamamatsu shizuoka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctakanashi bmc cancer page of lung cancer is one of the leading causes of cancerrelatedmortality worldwide radical resection is the standardtreatment for stage iii nonsmall cell lung cancer nsclc however the postoperative survival rate remainsunsatisfactory despite complete resection among patientswith nsclc who received complete resection experience local or distant disease recurrence thereforeadjuvant chemotherapy should be administered to improve survival after a radical surgery adjuvant chemotherapy has been shown to reduce therisk of death due to lung cancer recurrence [] nonetheless not all patients who underwent radical surgerybenefit from adjuvant chemotherapy because some ofthem are already successfully healed without adjuvantchemotherapy therefore patients highly at risk for recurrence who are likely to benefit from adjuvant chemotherapy should be identified for the efficient applicationof adjuvant chemotherapyadenocarcinoma is the most common histologicaltype of nsclc accounting approximately of allnsclc cases in lung adenocarcinomas severalprognostic factors obtained by histopathological evaluations of surgical specimens have been reported to datesuch as lymph node metastasis pleural invasion lymphatic vessel invasion [ ] blood vessel invasion[ ] adenocarcinoma subtype of micropapillary pattern and spread through air space stas [ ]however predicting lung cancer recurrence after radicalsurgery is still difficult because data on the direct relationship between conventional prognostic factors and recurrence are limited furthermore subjective judgmentsof conventional prognostic factors are considered to hinder accurate recurrence prediction and its retrospectivevalidation accordingly novel recurrence predictors withhigh objectivity are strongly expectedexampleprevious studies demonstrated that lipid metabolismalterations in cancer contribute to cancer cell proliferation and invasion [ ] and some lipids have beensuggested as prognostic factors in several cancer typesforthe number of phosphatidylcholinepc321 in recurrent cases of primary triplenegativebreast cancer tnbc is higher than in that of nonrecurrent cases and thereby pc is suggested as acandidate predictor for tnbc recurrence oleicacid attenuation is correlated with shorter progressionfree period in clear cell renal carcinoma with regard to lung cancer although nsclc is reportedly characterized by drastic changes in phospholipid profiles ascompared to the normal lung tissue and contains different lipid profiles according to the histologic subtypes no lipidomic approach to investigate the prognosticfactor for nsclc has been used based on these previous studies we hypothesized that lung adenocarcinomaswith high recurrence risk have different lipidomes fromthat of lung adenocarcinomas with low recurrence riskand specific lipids that can be considered as candidatesas novel predictive factors for recurrencein this study lipid species that can be considered as potential predictors for lung adenocarcinoma recurrence aftera radical surgery were identified by comparing lipidomes ofprimary lung adenocarcinomas between recurrent andnonrecurrent cases using liquid chromatographytandemmass spectrometry lcmsmsmethodspatients and tissue samplesretrospective frozen tissue samples of primary lungadenocarcinoma obtained from patients who underwentradical surgery from january to december athamamatsu university hospital were examined radicalsurgery was defined as complete resection performedwith lobectomy or pneumonectomy accompanied by systematic lymph node dissection at stage i or ii and ascomplete resection achieved by segmentectomy orwedge resection with or without lymph node samplingat stage i tissue samples of primary tumors were collected immediately after the resection and stored at °c after a rapid freezing in liquid nitrogen histopathological diagnosis was performed by experienced pathologists according to the world health anizationcriteria pathological staging was identified based on the8th edition ofthe tnm classification for lung andpleuraltumors patients were followedup withcomputed tomography ct of the body trunk and biochemicalantigencea every months during the first years thenevery months until more than years after the surgerywhen cea was elevated ¥ ngml without any ctfindings of recurrence head magnetic resonance imaging and systemic positron emission tomography wereperformed for the detection of brain metastasis or bonemetastasiscarcinoembryonicexamination ofin patient selection clinical records of these tissuesamples were retrospectively reviewed patients withpathological stage i or ii indicated for radical surgeryand with major histological subtypes of invasive adenocarcinoma lepidic papillary acinar or solid predominant were analyzed patients who received inductionchemotherapy or radiotherapy and those with other subtypes of adenocarcinoma were excludedthencases withoutand with recurrence wereassigned to nonrecurrent and recurrent groups respectively recurrence was defined as radiological imagingbased findings of distant or locoregional recurrencewithin years whereas no recurrence was defined as nofindings of distant or locoregional recurrence in ¥ yearsafter the radical surgery in the nonrecurrent group 0ctakanashi bmc cancer page of cases with followup period of years were excludedin the recurrent group cases with recurrence in theform of pleural dissemination were excluded assumingthe possible attribution with insufficient surgical marginfinally cases for recurrent and for nonrecurrentgroups were subjected for analysishistological evaluationparaffinembedded tissue blocks were sectioned at μmthick sections stained by hematoxylineosin he wereexamined for adenocarcinoma subtypesizelymph node metastasis and stas d2 stain wasused to evaluate lymphatic vessel invasion and elasticavan gienson stain to evaluate blood vessel invasion allhistologicalsections were reviewed by experiencedpathologiststumorchemicalsmethanol chloroform glacial acetate and ultrapurewater were purchased from wako pure chemical industries osaka japan the 12dilauroylsnglycero3pcavanti polar lipids alabaster al pc 120_120 wasused to calibrate standard lipid levelslipid extraction from the cancer tissueeach weight of the frozen tissue samples was measuredusing sartorius analytical lab balance cpa224s sartorius ag göttingen germany additional file supplemental table after the weight measurement modifiedblighdyer methods were performed for lipid extractiontissue samples were transferred into glass tubes and ml of methanol ml of chloroform and mlof m glacial acetate were subsequently addedthen mmol of pc 120_120 per mg of sampletissue was added and subsequently followed by 10minextraction at room temperature after the extraction ml of chloroform was added and vortexed sequentially ml of m glacial acetate was added andvortexed extracted samples were subjected to centrifugation at rpm for min extracted anic layerswere transferred into new glass tubes and were evaporated until completely dried using mivac duo lv genevacextracted lipid wasdissolved with μl of methanol and μl of the dissolved lipids were diluted again with methanol proportional to the weight of the original tissue samples so thatthe concentration of pc 120_120internal controlwill be as similar as possible among casesipswich england thelipid analysis by liquid chromatographytandem massspectrometry lcmsmsextracted lipids from collected frozen tissue sampleswere analyzed using q exactive¢ hybrid quadrupoleorbitrap¢ massanequipped withspectrometerelectrospray ionization source and connected to an ultimate system thermo scientific μl of the extracted lipid samples were injected and separated onacculaim c18 column mm à mm μmthermo scientific components of mobile phase awere as follows wateracetonitrilemethanol vvv mm ammonium formate and formic acidthe components of mobile phase b were as followsacetonitrileisopropanol vv mm ammonium formate and formic acid for elution the flow ratewas set at μlmin a set of linear gradient startingat solvent b was used and linearly increased to b in min maintained at b until minthen decreased linearly to b from min to min and finished with b for the last min theoverall run time was min ms instrument conditionswere as follows sheath gas flow rate auxiliary flowrate sweep gas flow rate capillary temperature °c slens rf level probe heater temperature °c and spray voltage of kv in positive mode and kv in negative mode fullms mode conditions forquantification were as follows ms scan range resolution agc target à and maximum injection time was ms for identification top datadependent ms2 method with a resolution of was used the agc target was à and themaximum injection time was ms stepped normalizedcollision energies of and for the positivemode and and for the negative mode wereapplied spectral data were acquired in the mz range of mz using an xcalibur v30 software thermoscientifictolerance ppmlipid identification and quantificationlipidsearch¢ software version mitsui knowledgeindustry tokyo japan was used to identify and quantify lipid species parameter settings for identificationwere followings database hcd retention time min search type product_qex precursor tolerance ppm and productidentificationquality filters of a b and c were used quantificationwas performed at mz tolerance of ± with retentiontime range from min to min alignment of theidentified lipid species among cases was performedwith retention time tolerance of molecules that areannotated as redundant lipid names with different calculated mz and retention times were regarded as independentisomersinadditional file duplicationannotatedasdata processingtrend analysis between the nonrecurrent and recurrentgroups was performed by comparing the average totallipid level between the two groups and principal 0ctakanashi bmc cancer page of component analysis pca intensities of lipids recordedin the xcalibur v30 software and monoisotopic peakarea values of lipid species identified by lipidsearch¢software were normalized by dividing with the areavalues of internal control pc 120_120 the total lipidlevel of each case was defined as an accumulation ofnormalized intensities of lipids normalized area valueswere subjected for pcafor respective lipid species pvalues were calculatedusing the student ttest to compare area values betweenthe two groups to screen candidate lipids for recurrence prediction lipidomes were compared between thenonrecurrent and recurrent groups by describing volcano plots with log10 pvalue for vertical axis andlog2 folding change for horizontal axis the foldingchange for a lipid was defined as an average area valueof the recurrent group divided by that of the nonrecurrent group significance was determined at pvaluesof folding change of ¥ or statistical analysisdemographic information and associations with clinicalcharacteristics were evaluated using the fisher exact testcategorical variables or the mannwhitney utest forcontinuous variables the student ttest was used tocompare the average totallipid amounts of the nonrecurrent and recurrent groups and to describe volcanoplots recurrentfree survival rfs was determined asthe time from operation until the first disease recurrenceor death survival curve was described using thekaplanmeier method the optimal cutoff values todiscriminate the two groups were determined using thereceiver operating characteristic roc curve analysisthe area under the roc curves aucs were calculatedto validate the discrimination abilities of candidate lipidsspearmans rank correlation analysis was used to validatethe correlation among candidate lipid predictors allstatistical analyses except for the ttest were performedusing r the r foundation for statistical computingvienna austria version the student ttest wasperformed with ttest of excel¢ microsoft redmond usa pvalues of were considered assignificantresultsclinicopathological characteristics of patient cohortclinicopathological characteristics of patients are shownin table in this study cohort tissue samples from nonrecurrent and recurrent cases were analyzedamong the characteristics of these two groups differences in pathological stage p lymph node metastasis p and blood vessel invasion p were statistically significant the and 2year rfs rateof the recurrent group was and with median rfstime of range monthsrespectivelyfile supplemental fig the medianadditionalfollowup time ofthe nonrecurrent and recurrentgroups was range and range months respectivelytrend analysis between the nonrecurrent and recurrentgroupsthe frozen tissue samples were subjected to lcmsms and the total lipid level of cases was calculated byaccumulating normalized intensities of lipids notablythe average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp fig a total of lipid species wereidentified and quantified by analyzing the mass spectraldata using a lipidsearch¢ software the full list of identified lipid species is presented as additional file which were also subjected to pca the pca plot didnot show clear separation between the recurrent andnonrecurrent groups however the recurrent group exhibited partial separations between the first three principal components additional file supplemental fig these results suggested differences of lipidome betweenthe recurrent and nonrecurrent groups which urged usto screen lipids to distinguish the two groupsscreening of candidate lipids for recurrence predictionto screen lipids with different levels between the twogroups volcano plots of the identified lipids were described first and lipidomes between the nonrecurrentand recurrent groups were compared fig the volcano plotidentified lipid species with relativeamounts significantly different between the two groupsfolding change ¥ or pvalues thenumber of lipids that increased and decreased in the recurrent group was and respectively these increased or decreased lipid species consisted of varioushead groups additional file increased lipid speciesshown in red decreased lipid species shown in greenthen based on prominent distributions of the volcanoplot we narrowed the candidate lipids increased inthe recurrent group to the following molecules fig biotinylbluephosphoethanolamineceramidecerd420 sphingomyelin smd351 cerd180_240pc monoether phosphatidylcholine mepc346echolesterol ester che241 mepc 408e and che20 as for the lipids that decreased in the recurrent groupthe following four molecules were annotated fig bluearrows pointing to green plots monohexosylceramidehex1cert421 otriglyceride tg150_140_140pc 182_182 and lysophosphatidylcholine lpc120biotinylpe303arrowsplotspointingtoredthe relative amounts of these lipid species were evaluated with their distributions by comparing the two 0ctakanashi bmc cancer page of table clinicopathological characteristics of the nonrecurrent and recurrent groupscharacteristicsmedian age rangenonrecurrent n gender malefemalesmoking history pathological stage iiimedian tumor size mm rangeadenocarcinoma subtypelepidicpapillaryacinarsolidlymph node metastasis pleural invasion lymphatic vessel invasion blood vessel invasion micropapillary component spread through air space driver gene mutationegfr alk surgical procedurelobectomywedge resectionadjuvant chemotherapyindication stage ia3iibreceivedrecurrent stylelocoregionaldistant recurrent n pvalueabbreviations alk anaplastic lymphoma kinase egfr epithelial growth factor receptroups fig 3a and b in all tested lipids distributionsbetween the two groups were well separated enough toestablish the cutoff values whereas only few markedoutliers were foundwe next calculated the cutoff values and auc of these lipids to evaluate their discrimination ability for diseaserecurrence and the following final candidates with topthree auc were selected smd351 cerd420 and tg 150_140_140 table respective lipidspecies can be found in additional file with the followingidentical numbers smd351 cerd420 andtg 150_140_140 these three final lipid candidates were annotated as the following ions [smd351 h] [cerd420 hcoo] and [tg 150_140_140 nh4] in the lipidsearch¢ software additional file msms for [smd351 h] [cerd420 hcoo]and [tg 150_140_140 nh4] demonstrated production peaks corresponding to phosphocholine severalfragments compatible with fragmentation of cerd42 with concomitant oxidation reaction two fragmentsproduced by neutralfatty acid fa140 orfa respectivelyadditional file supplemental fig consequentlythe annotations ofthe final candidates by lipidsearch¢ software were consistent with the results ofmsmsfrom tg 150_140_140loss ofamong these three candidate predictors smd351was found to be positively correlated with cerd420spearmans rank correlation coefficient[rs] p tg 150_140_140 was inversely correlated with smd351 rs p and tg150_140_140 was weakly inversely correlated withcerd420 rs p additional file supplemental fig 0ctakanashi bmc cancer page of conventionalpathologicalvalidation of recurrence prediction ability among thefinal lipid candidatestable shows the sensitivity specificity and accuracyof the final candidate lipid predictors compared withfactorstheprognosticlymph node metastasis and blood vesselinvasionwhich were identified as significant recurrent factorsin this cohort sensitivity of all three candidate lipidpredictors is superior to that of lymph node metastasis patients with lymph node metastasis all of themwere hilar or lobar lymph node metastasis corresponded to those in stage ii among the recurrentgroup in this study cohort half of the study population had stage i whereas the other half had stage iias lymph node metastasis can be detected amongstage ii cases the sensitivity of lymph node metastasiswas consequently lower than those of three candidatelipid predictors which detected both stage i and stageii hencethese three predictors were superior tolymph node metastasis for patient screening whencomparing the candidate lipid predictors and bloodvesselshowed predictionto those of blood vesselabilities higher or equalinvasion only smd351fig comparison of total lipid levels between the recurrent andnonrecurrent groups the average total lipid level of the recurrentgroup was times higher than that of the nonrecurrentgroup p fig volcano plots of identified lipid species each plot represents a lipid species to be identified the relative amount of lipid speciesred plots were increased fc ¥ right side of in the horizontal axis pvalue in vertical axis and that of lipid species greenplots were decreased fc left side of in the horizontal axis pvalue in vertical axis in the recurrent group nineincreased lipids showing prominent distributions and all decreased lipid species were annotated for candidate predictors blue arrowsabbreviations cer ceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepcmonoether phosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine sm sphingomyelin tg triglyceride 0ctakanashi bmc cancer page of fig comparisons of relative amount distributions between the nonrecurrent and recurrent groups are shown for increased a and decreasedb lipid species in the recurrent group boxplots show the upper percentile upper quartile median lower quartile and lower percentilemaximum and minimum values are shown in dots pvalues for significance and fcs are presented for each lipid species abbreviations cerceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepc monoetherphosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine tg triglycerideinvasion in allvalidation points therefore wepropose smd351 as the most hopeful candidate forrecurrence predictiondiscussionin this study candidate lipid predictors for lung adenocarcinoma recurrence after a radical surgery were retrospectively screened and smd351 was found as themost prominent predictor showing that the predictionability was superior to that of conventional pathologicalprognostic factors in this small cohortthe average total lipid level was significantly high inthe recurrent group in this study furthermore thenumber ofincreased lipid species was considerablyhigher than that of decreased lipid species in the recurrent group these results were consistent with that of 0ctakanashi bmc cancer page of table auc rank of candidate lipid predictors determined byroc curverankcutoff valuespeciessmd351cerd420tg150_140_140cerd180_240pc182_182che241pc412biotinylpe303lpc120hex1cert421 omepc408eche201mepc346eauc ci lipids with top three auc were selected as final candidate predictorsboldfaced notationsabbreviations auc area under the roc curve ci confidential interval rocreceiver operating characteristicprevious studies that showed an accelerated lipid synthesis in cancer cells contributing to tumor phenotypes such as cellular membrane building stimulationof signaling pathways for growth and proliferation orsurvival under hypoxic conditions by supporting glycolysis [ ] increased total lipid level in the recurrent group may be biologically plausible because theaggressiveness may be supported by accelerated lipidsynthesisthe number of smd351 and cerd420 two of finalcandidate predictors were increased in the recurrentgroup sm and cer are major bioactive components oflipid rafts on the cellular membrane sm is synthesized from cer by sm synthase sms which transfersthe phosphocholine head group from phosphatidylcholine to cer and results in concomitantly producingtable comparison of sensitivity specificity and accuracyamong the three final candidate predictors and conventionalhistopathological prognostic factorspredictors for recurrencecandidate lipid predictorsspecificitysensitivityaccuracysmd351cerd420tg150_140_140pathological prognostic factorslymph node metastasisblood vessel invasionsmd351 showed the most excellent prediction abilityabbreviations cer ceramide sm sphingomyelin tg triglyceridediacylglycerol dag sm reconversion to cer is catalyzed by sphingomyelinase smase increased smabundance and sms activity have been reported to playa critical role in cell proliferation and survival in severalcancer types [] with regard to lung cancer metabolic changes in sphingolipids are suggested to correlatewith chemoresistance phenotype and the total smlevel in cancer tissues is reportedly lower than that ofthe normallung tissue in patients with nsclc this is speculated in the report that decreased sm abundance in lung cancer tissues may be attributable to highconsumption of serine precursor by highly proliferatingcancer cells cer accumulation in the lungs has beensuggested to participate in both cell apoptosis andtumorigenesis under cigarette smokeinduced oxidativestress taking together these knowledge and significant positive correlation between smd351 h andcerd420 in this study increased synthesis flow of certoward sm in the recurrent group was suggested actually significant increase on the total sm p leveland increased tendency on total cer p anddag p levels in the recurrent group were observed in this study cohort additional file supplemental fig this result supports the suggestion ofstrong synthesis flow of cer toward sm the sm andcer levels were not compared between the tumor tissuesand normal lung tissues in this study because normallung tissue samples were lacking nonetheless increasedsmd351 and cerd420 in the recurrent group in thisstudy is consistent with previous studies [ ]based on the following explanation among lung adenocarcinomas with high sm and cer consumption casesthat can maintain increased sm and cer synthesis havehighly aggressive phenotypes resulting in recurrencedecreased tg 150_140_140 in the recurrent groupwas also included in the final candidate predictors although tg abundance in the lung cancer tissue has not yetbeen explored to date tg level in colon cancer is reportedto be lower as the disease progresses suggesting that energysupply for colon cancer with higher degree of malignancymay depend on tg hydrolysis inconsistent with theprevious study the total tg level in this study revealedno significant difference between the nonrecurrent and recurrent groups p possible explanation for decreased tg 150_140_140 in the recurrent group is thataggressive recurrent lung adenocarcinoma that may preferably consume specific tg species for energy supplythe difficulty of predicting lung cancer recurrenceusing histopathological prognostic factors may be partlyattributed to subjective judgement in addition althoughthe degree of histopathological prognostic factors widelyvaries their judgements have been performed qualitatively [ ] thereby these methods may hinder accurateretrospectiverecurrence prediction and its 0ctakanashi bmc cancer page of between representativerecurrentimages of papillarytype adenocarcinomavalidation conversely excellent prediction ability ofsmd351 that is superior to histopathological factorswas considered for its high objectivity and quantitativevalues actuallyit was difficult to predict recurrentprognosis objectively from the conventional histopathologicalthemost popular tissue subtype with no significant differenceand nonrecurrent cases whereas the mass spectrum intensitiesof [smd351 h] were markedly higher in the recurrent case to help recurrence prediction additional file supplemental fig furthermore as high smd351level was detected in all recurrent cases including stagei and stage ii cases with high specificity and accuracysmd351 was considered to be widely applicable for recurrence prediction in postoperative patients whounderwent radical surgeryseveral limitations in this study should be acknowledged first this retrospective study is performed on asmall sample size due to difficulty of obtaining frozensurgical specimens with clinical information that meetour inclusion criteria thereby verifying the reproducibility of using other validation cohorts was difficult thusidentified lipid predictors did not exceed above the candidate levels and further large cohort studies should beconducted to validate candidate predictors identified inthis study as rigid predictors for lung adenocarcinomarecurrencebecause a large number of candidate lipid species species relative to the small number of samplesize cases were screened for candidate predictorsone candidate that shows nearperfect discriminationability can be bound to be identified third adjacentnormal lung tissue samples were lacking hence the difference between the abundance of the identified candidate lipid predictors in the normal lung tissue of therecurrent group and that of the nonrecurrent groupwas not able to be compared fourth because the nonrecurrent group in this study included five cases that received adjuvant chemotherapy the nonrecurrent groupmay possibly include the recurrence highrisk casesamong them recurrence might be prevented by adjuvantchemotherapy moreover the nonrecurrent group inthis study included two cases with recurrence predictionpositive for smd351 additional file supplementalfig among the two cases one patient received adjuvant chemotherapy and the other did not the formercase may be considered as highly at risk for recurrencewhich was prevented by adjuvant chemotherapy thelatter may be an exceptional case that cannot be ruledout by smd351 fifth because lcmsms is not auniversal examination in the clinical field examining alarge number of surgical specimens for recurrence prediction using lcmsms is difficult to utilize thefindings of this study in a clinical field lipid predictorsshould be replaced with other molecules that can be examined by universal methods such as immunohistochemistry of sms or smase involved in the smmetabolism additionallyin thisstudy included histopathological type of adenocarcinomaonly as a topic for future study squamous cell carcinoma a major histological subtype behind adenocarcinomarecurrent predictorsshould be explored forthrough the lipidomic approachthe sample cohortswe propose that smd351 is a hopeful candidate predictor for lung adenocarcinoma recurrence after a radical surgery our findings provide novel insights on themechanisms oflung adenocarcinoma recurrence andcan contribute to the development of precise recurrenceprediction and qualified postoperative therapeutic strategy for lung adenocarcinomasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020073061additional file supplemental table weights of the frozen tissuesamples each weight of the frozen t | 0 |
" deregulated circular rnas circrnas are associated with the development of cancer and therapyresistance however functional research of circrnas mostly focus on potential mirna or protein binding and morepotential regulation of circrna on host gene dna in cancers are yet to be inspectedmethod we performed total rna sequencing on clinical breast cancer samples and identified the expressionpatterns of circrnas and corresponding host genes in patient blood tumor and adjacent normal tissues qpcrnorthern blot and in situ hybridization were used to validate the dysregulation of circrna circsmarca5 a series ofprocedures including rloop dotblotting dnarna immunoprecipitation and mass spectrum etc were conductedto explore the regulation of circsmarca5 on the transcription of exon of smarca5 moreoverimmunofluorescence and in vivo experiments were executed to investigate the overexpression of circsmarca5with drug sensitivitiesresults we found that circrnas has average higher expression over its host linear genes in peripheral bloodcompared to adjacent normal tissues circsmarca5 is decreased in breast cancer tissues contrary to host genesmarca5 the enforced expression of circsmarca5 induced drug sensitivity of breast cancer cell lines in vitro andin vivo furthermore we demonstrated that circsmarca5 can bind to its parent gene locus forming an rloopwhich results in transcriptional pausing at exon of smarca5 circsmarca5 expression resulted in thedownregulation of smarca5 and the production of a truncated nonfunctional protein and the overexpression ofcircsmarca5 was sufficient to improve sensitivity to cytotoxic drugs our results revealed a new regulatory mechanism for circrna on its host gene and provided evidencethat circsmarca5 may serve as a therapeutic target for drugresistant breast cancer patientskeywords breast cancer circrna dna damage repair rloop host gene correspondence kechenhusteducn chewhueducnleiweifrhotmailcom xiaolong xu jingwei zhang yihao tian and yang gao contributed equallyto this work3department of urology tongji hospital tongji medical college huazhonguniversity of science and technology wuhan china1school of basic medical sciences wuhan university wuhan hubeichinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cxu molecular cancer page of introductioncircular rnas circrnas are novel rnas that havebeen ubiquitously discovered in many species by highthroughput sequencing in recent years [ ] circrnasare generated by the backsplicing of intronic exonic orintergenic regions circrnas are resistant to rnase rand the stability of their structures makes these molecules ideal candidates for disease extensive studieshave revealed that dysregulated circrnas are involvedin the development of various cancers in gastric cancercircrnassuch as circpvt1 circlarp4 has_circ_ and circ_100269 have been shown to play arole in promoting tumor growth and their expression iscorrelated with high tnm stage and poor prognosis []in colon and hepatic carcinoma cirs7 promoted tumordevelopment and progression by activating the egfr andpi3kakt pathway [ ] circrnas such as circkif4ahsa_circ_0001944 hsa_circ_0001481 and circrna_0025202have been implicated in molecular typing brain metastasisand drug resistance in breast cancer [] although greatprogress has been made the roles of circrna and relevantmolecular mechanisms remain largely unknownprevious studies have shown that circrnas exert theirfunctions in different ways as noncoding rnas circrnas regulate the expression of other genes by servingas sponges for microrna and rnabinding proteins[ ] in addition some circrnas have been shownto be translated into functional proteins [ ] inaddition circrnas have also been shown to directlyinteract with the genomic dna of the host gene inplant which results in altered parent gene expression however the interaction of circrnas and hostgene dna were less studied in human cancerssmarca5 is a member of the swisnf complex withatpdependent chromatin remodeling activity []in the process of dna damage repair smarca5 isinvolved in chromatin remodeling in dna damageregions providing a structural basis for the recruitmentof dna damage repair factors [ ] in tumorssmarca5 is highly expressed in hepatic carcinoma andprostate cancer and its expression levelis inverselyrelated to tumor radiosensitivity [ ]in this study we established circrnas have averagehigher expression than their host genes in peripheralblood comparing to tissues then we identified acircrna derived from smarca5 circsmarca5 issignificantly decreased in breast cancer cell lines andbreast cancer samples differentto previous worksrevealing circsmarca5 can also function as a competing endogenous rnas by binding with mirnamoleculesour mechanism explorationdisplayed circsmarca5 is involved in regulating dnarepair capacity by binding exon dna directly andfurther functionalinvestigation of this circrna may[]contribute to the therapeutic implications for cytotoxicdrugresistant breast cancer patientsresultsidentification of expression of circrnas in breast cancerwe performed high throughput sequencing on tumort and adjacent normal tissue an and peripheralblood b of six breast cancer patients total rna withrrnadepleted library wereconstructed and thencircrnas expressed in those samples were identifiedcompared to tumor and adjacent normal tissue we observed average higher circscore expression of circrna linear host genes in blood than both tumor andadjacent normal tissue in all circrnas which wereexpressed across all six patients we observed averagecircscores from to in blood which is higherthan tumor to and adjacent normal tissue to in six patients fig 1a this result indicated average higher expression of circrnas than theirhost genes in peripheral blood comparing to tissueswhich might contribute to the exploration of diagnosticbiomarker for breast cancer we then selected sixcircrnas with high circscores average to in patients and performed further experimental validation in patients realtime pcr results establishedtwo circrnas circhipk3 and circsmarca5 weresignificantly differentially expressed between tumor andadjacent normal tissue fig 1b and figure s1a especially circsmarca5 was lower expressed in tumorsamples and less studied in previous work furthermorethe ratio of circtolinear expression of circrna linearhost genes of circsmarca5 in blood sample of healthvolunteers were significantly higher than those of breast cancer patients p fig 1c and figure s1bwe nextcirctolinear ofcircsmarca5 and clinical relevance in patients withbreast cancer and observed significant difference in thedistribution of the patients according to pathologic t p table s1 together these results indicating thepotential function and candidate biomarker attributes ofcircsmarca5 in breast cancerexamined theratio ofand functionallyinvestigatefound thatto characterizecircsmarca5 we firstly detected the expression of circsmarca5 in cell lines circsmarca5 is derived fromthe backsplicing of exon and exon of smarca5fig 1d as expected endogenous circsmarca5 butto rnase r digestionnot premrna was resistantfig 1dthe ntcircsmarca5 was further confirmed by northern blotassaycircsmarca5 was mainly present in the nucleus whereasits parent mrna was present exclusively in the cytoplasm as evidenced by qpcr northern blotting andrna in situ hybridization fig 1fh and figure s2fig 1e furthermore wethe existence ofin addition 0cxu molecular cancer page of fig see legend on next page 0cxu molecular cancer page of see figure on previous pagefig identification of circrnas in breast cancer a heatmap of circscore fbpcircfbplinear in tumor t adjacent normal tissue an and bloodsample b from six breast cancer patients b expression of six circrnas with high circscore were validated by rtqpcr assay in breast tumor andadjacent normal tissue represents p circrnas ids are according to circbase through their genomic coordinates c the ratio of circtolinear ofcircsmarca5 in blood sample of breast cancer patients and health volunteers total rna from blood sample of breast cancer patients and healthvolunteers was extracted and detected by rtqpcr the expression level was normalized with βactin as reference p was consideredstatistically significant d schematic illustration showing the genomic region of circsmarca5 derived from exons and of the smarca5 geneconvergent gray and divergent black primers were designed to amplify the linear or backsplicing products upper total rna from mcf7 cellswith or without rnase r treatment was subjected to rtpcr lower and further validated by sanger sequencing right e northern blot using ajunctionspecific probe or an exon probe showing the endogenous existence of circsmarca5 and smarca5 mrna from mcf7 cells with orwithout rnaser treatment r or r the bp marker indicates the smarca5 fulllength transcript transcribed in vitro the bp markerindicates exon and exon of smarca5 transcribed in vitro f the nucleus and cytoplasm mrna of mcf7 were extracted and smarca5 andcircsmarca5 expression levels were quantitated by rtpcr gapdh and hu6 serve as internal references of the cytoplasm and nucleus respectively indicates p g the nucleus and cytoplasm mrna of mcf7 were extracted smarca5 and circsmarca5 were examined by northernblotting and the smarca5 exon probe was applied in this experiment h subcellular localization of circsmarca5 and smarca5 in mcf7 cellsthe signals were examined by indirect rna fish and confocal microscopy the nucleus was counterstained with dapi the circsmarca5 probe waslabled by biotin while the smarca5 probe was labled by dig they were stained with red and green fluorescent secondary antibodies respectively ithe expression of circsmarca5 detected by northern blot mdamb231 bt474 mcf7 skbr3 are breast cancer cell lines mcf10a are normal breastcell line n1n2n3n4n5 are adjacent normal tissues t1t2t3t4 are breast cancer tissues indicates p next we examined the expression of circsmarca5 invarious breast cancer cell lines mcf7 skbr3 bt474mdamb231 and immortalized but nontransformedbreast epithelial cells mcf10a as well as in adjacentnormal tissues and breast cancer tissues northern blotresultsthe expression levels of circsmarca5 in mcf10a and normal adjacent tissuesare higher than breast cancer cell lines and cancer tissues fig 1i these results indicated that circsmarca5is downregulated in breast cancer tissues and cellsrevealed thatsequencecircsmarca5 decreases the expression of smarca5 incancer cellsto clarify the mechanisms of circsmarca5 we investigated its effects on the expression of its parent genesmarca5 the expression levels of circsmarca5 andsmarca5 were detected by the primers of junctionsequence and exonsrespectivelyknockdown of circsmarca5 increased both mrnaand protein levels of smarca5 while converselycircsmarca5 overexpression decreased smarca5levels fig 2ac and figure s3 consistently the proteinof smarca5 was high expressed in breasttumorsamples as compared with the corresponding controlsfigure s4 moreoverthe ratio of circtolinear ofcircsmarca5 was significantly lower in breast andrenaltumor tissue than the corresponding adjacenttissue specimens fig 2de and figure s1c besides asignificant negative correlation was also found betweencircsmarca5 and smarca5 expression in various celllines and primary cancer tissues fig 2df and figures5 which corroborates our observation that circsmarca5 decreased the expression of smarca5 incancer cellscirsmarca5 terminates the transcription of smarca5 atexon we further investigated the mechanism of circsmarca5in regulating the expression of smarca5 interestinglywe found that the overexpression of circsmarca5 indeed decreased the expression of smarca5 exons but had minimal effects on the expression of exons fig 3a next we designed a primer location in exon for the amplification of ² cdna ends by rapid amplification of cdna ends race pcr fig 3b left as shownin fig 3b smarca5 can give rise to multiple isoformsimportantly we found a decrease in a band of bpupon circsmarca5 overexpression while an bpband displayed the opposite phenomenon fig 3b rightsanger sequencing showed that the bp band andthe bp band are derived from fulllength and truncated mrna exons to respectively of the smarca5 gene fig 3c consistent with the race resultsnorthern blot assay further demonstrated that ectopiccircsmarca5 expression decreased smarca5 levelsand promoted truncated mrna levels fig 3d the observations gathered thus far have led us to hypothesizethat circsmarca5 prevents transcription from exon of smarca5 indeed chip analysis indicated that thebinding of pol ii to exons of smarca5 was higherthan that to exons fig 3e left and the ectopic expression of circsmarca5 decreased the binding of pol iito exons of smarca5 fig 3e right to furtheraddress whether circsmarca5 could terminate the transcriptional elongation of smarca5 we cloned a series ofexons of smarca5 in a luciferase plasmid reporterfig 4a upper the transient transfection of these luciferase reporters containing the exon sequence revealed that luciferase activity was significantly decreasedwhen circsmarca5 was overexpressed fig 4a lower 0cxu molecular cancer page of fig circsmarca5 decreases the expression of smarca5 in cells a generation of circsmarca5knockdown and circsmarca5overexpressingcells mcf7 cells were infected with lentiviruses expressing shrna against circsmarca5 shcircsmarca5 three different oligonucleotides orcircsmarca5 plcdhcircsmarca5 rtqpcr was performed to evaluate the expression of circsmarca5 gapdh was used as an internal controlb rtqpcr showing the levels of circsmarca5 and smarca5 in mcf7 cells stably expressing shnc shcircsmarca5 plcdhcir control orplcdhcircsmarca5 c western blot showing the levels of smarca5 in mcf7 cells stably expressing shnc shcircsmarca5 plcdhcircontrol plcdhcircsmarca5 plcdhcircsmarca5δ without splicinginducing sequence gapdh was used as an internal control df theratio of circtolinear of circsmarca5 in tumor tissue were significantly lower than normal tissue in breast cancer samples d and rcc samplese p a negative correlation between circsmarca5 and smarca5 expression was observed in breast cancer samples d rcc samplese and various cell lines fto further confirm the effect of circsmarca5 on thetranscriptional elongation of smarca5 we insertedexons of smarca5 between dsred and egfp as indicated fig 4b upper the egfp level was significantly decreased by circsmarca5 when exons were present fig 4b lower we further investigatedthe role of circsmarca5 in the regulation of smarca5 at the protein level as expected circsmarca5overexpression downregulated the protein levels ofsmarca5 and upregulated truncated smarca5δsmarca5 protein levels fig 4c and figure s6which was confirmed by mass spectrometry fig 4dmoreover we found that δsmarca5 is more susceptible to proteolysis by the proteasome than smarca5 fig 4e together these results show the roleof circsmarca5 in the termination of transcriptionalelongation at exon of smarca5circsmarca5 can form rloops with its parent geneto further dissect the mechanism of smarca5 transcriptional termination mediated by circsmarca5 weinvestigated whether circsmarca5 can bind genomic 0cxu molecular cancer page of fig cirsmarca5 terminates the transcription of smarca5 at exon a rtqpcr analysis of the expression of smarca5 in mcf7 cells using aseries of paired primers indicates p b rapid amplification of cdna ends race pcr analysis of smarcac5 transcripts the pcrproducts were readily identified by agarose gel electrophoresis each set of samples was repeated three times c sanger sequencing of twotranscripts of smarcac5 that are regulated by circsmarca5 overexpression d northern blotting using the junctionspecific probes for exons and to show the expression levels of the transcripts of smarcac5 mrna from mcf7 cells stably expressing control vector or plcdhcircsmarca5 circoe e circsmarca5 prevents transcription from exon of smarca5 chipseq analysis showing that the binding of pol ii toexons of smarca5 was higher than that to exons chipqpcr showed that the ectopic expression of circsmarca5 decreased thebinding of pol ii to exons of smarca5smarca5 dna to form an rloop dotblotting withrloopspecific s96 antibody supported our hypothesisthat circsmarca5 can bind exons of smarca5genomic dna fig 5a additionally we performeddnarna immunoprecipitation drip qpcr and confirmed the interaction between circsmarca5 andexons pretreatment with rnase h ablated thisinteraction confirming that the interaction is rloopspecific fig b and figure s7 the interaction of circsmarca5 with the dna of smarca5 was directlyverified by fluorescence in situ hybridization fig 5cconsistent with previous findings dotblotting ofthe genome without rna digest revealed that the binding of circrna to genomic dna may be widely presentin cancer cells fig 5d we next determined the specificsequence of exons required for rloop formationa series of fragments from exons were hybridizedwith circsmarca5 for the dotblotting assay as shownin fig 5e the bp fragment of the ² end of exon plays important role in interacting with circsmarca5moreoverthe secondary structure of circsmarca5was determined by the software mfold whichrevealed the sequence ²aacaaaauugggaaagaugaaaugcuucaaau3² from the ² end of exon located in the loop region of circsmarca5 fig 6awe thus hypothesized that this sequence might play akey role in mediating the circsmarca5dna interaction to this end we synthesized the wildtype andmutant phosphorylated dna fragments ant andantmut respectively corresponding to this sequencefig 6b dotblotting demonstrated that wildtypeoligonucleotides ant can bind to circsmarca5 but 0cxu molecular cancer page of fig see legend on next page 0cxu molecular cancer page of see figure on previous pagefig cirsmarca5 blocks the transcription of smarca5 and promotes the generation of a truncated smarca5 protein δsmarca5 aschematics of luciferase reporter constructs containing the smarca5 exon sequence as indicated upper the smarca5 exon sequenceplays an important negative role in mediating the effect of circsmarca5 overexpression on luciferase activity lower b schematics offluorescence reporter constructs containing the smarca5 exon sequence as indicated upper mcf7 cells were transiently transfected withthese fluorescence reporters along with or without circsmarca5 cooverexpression after transfection for hours the reporter transcriptionactivities were measured by flow cytometry assay c circsmarca5 overexpression downregulated the protein levels of smarca5 whileupregulating truncated smarca5 δsmarca5 protein levels mcf7 cells stably overexpressing circsmarca5 or control cells were treated withdmso or mg132 western blot analysis was performed using an antibody targeting the nterminus of smarca5 to evaluate the expression ofsmarca5 and δsmarca5 gapdh was used as an internal control d the δsmarca5 protein was identified by mass spectrometry and detectedsmarca5 peptides were showed in the map the redlabeled portion is the amino acid sequence of the translated defective transcript e mcf7cells expressing flagsmarca5 and flagδsmarca5 were treated with cycloheximide chx μgml the cell lysates were subsequentlyharvested at sequential time points or h after treatment and then the cell lysates were immunoblotted with antiflag or antiactin antibodymutant oligonucleotides antmut cannot bind to circsmarca5 fig 6c as expected dripqpcr showedthat ant inhibited circsmarca5 binding to the dnaat exons whereas antmut had no effect on thisinteraction fig 6d furthermore the transfection ofant prevented the decrease in smarca5 proteinlevels in mcf7 cells stably expressing circsmarca5whereas antmut had no effect on smarca5 proteinlevels fig 6e importantly the mutation of the keysequence in circsmarca5 impaired the interactionwith its parent gene which was confirmed by dotblotting and dripqpcr assays fig 6fh and figures8 unlike circsmarca5 circsmarca5mut had littleeffect on smarca5 protein levels fig 6i theseresults suggested that circsmarca5 formed rloopswith its parent gene to inhibit the expression of smarca5 in cancer cellscircsmarca5 inhibits dna damage repair functionto explore the roles of circsmarca5 in cancer progression we overexpressed and depleted circsmarca5in mcf7 cells by lentiviral vectors and then examinedthe effect of circsmarca5 on cell proliferation migration and apoptosis however the results showed thatboth overexpressed and depleted circsmarca5 had noeffect on these three activities figure s9 previousstudies have indicated that smarca5 plays an important role in regulating the dna repair process and main[ ]taining theconsistent with previous reports smarca5 overexpression improved dna repair capacity and reducedthe expression of chk1 and chk2 after dna damagerepair figure s10a given that circsmarca5 canpromote the production of the truncated δsmarca5protein we tested whether the truncated protein is alsofunctional the overexpression of flagδsmarca5had a minimal effect on the expression of chk1 andchk2 after dna damage repair figure s10b suggesting that δsmarca5 isa nonfunctional proteinproduct we next assessed whether circsmarca5 canthe genomestability ofshowedlowerthan thatsignificantlyformation assaysaffect the function of dna damage repair capacitycck8 and clonerevealed thatcircsmarca5 overexpression increased sensitivity tocisplatin or bleomycin in mcf7 cells fig 7a b nextmcf7 cells were treated with the indicated concentration of cisplatin or bleomycin for h and then thedna damage was evaluated by single cell gel electrophoresis scge at and h mcf7 cells expressingcircsmarca5repaircapacity than did control cells fig 7c in paralleldna damage was examined after h of treatmentwith cisplatin or bleomycin by using an antiγh2axantibody consistent with the scge results the γh2axin mcf7 cells expressing circsmarca5 wassignalsignificantly higherin mcf7 cells asevidenced by immunostaining fig 7d consistentlycisplatin significantly enhanced the levels of dnadamage response proteins chk1 and chk2 in mcf7cellsexpressing circsmarca5 fig 7e whereasseveral key cellcycle genes were reduced specificallyupon circsmarca5 overexpression fig 7f to testwhether circsmarca5 rloop formation is necessaryfor its dna repair function we transfected ant orantmutinto circsmarca5expressing cells thescge assay and γh2ax measurement showed thatant significantly enhanced the dna repair capacitywhile antmut had no effect on this activity fig 7gand figure s11 furthermore ant significantlydecreased the degree of colocalization between circsmarca5 and its cognate dna locus figure s12in addition unlike circsmarca5 the overexpressionof circsmarca5mut had little effect on the dnarepair rate fig 7h and figure s13a b next we determined whether smarca5 could mediate the effects ofcircsmarca5 in preventing dna damage repair asshown in fig 7i the γh2ax signal was much lower incircsmarca5expressing cellscomplemented withsmarca5 than that in cells expressing circsmarca5alone as expected δsmarca5 could not rescue theinhibition of dna damage repair function induced by 0cxu molecular cancer page of fig see legend on next page 0cxu molecular cancer page of see figure on previous pagefig circsmarca5 interacts with its site of transcription a circsmarca5 interacts with the exon sequence of the smarca5 locus a seriesof exon dna fragments were hybridized with circsmarca in vitro the dnarna hybridization strength was quantified by dotblot with rloopspecific s96 antibody hybridization stringency was altered by decreasing ionic strength mm nacl b dripqpcr analysis of the exon sequence of smarca5 to detect the association of circsmarca5 in mcf7 cells rnase htreated andor dripqpcr analysis of the exon sequence as a control c circsmarca5 partially localized at its site of transcription double fish of circsmarca5 red and its parent dnaregion green the nucleus was stained by dapi d dotblot of rloops in mcf7 cell genomic dna preparations treated with dnase i rnase hor rnase r the dnarna hybrids in genome dna were analyzed by s96 antibody e mapping of the rloop formation region of circsmarca5a series of exon deletion mutants were hybridized with circsmarca5 for the dotblotting assay the dnarna hybridization intensity wasanalyzed by dotblot with an s96 antibody targeting the dnarna hybrid strand hybridization stringency was altered by decreasing ionicstrength mm naclcircsmarca5 figure s13c d moreover the overexpression of smarca5 could significantly rescue thegrowth defects of cells expressing circsmarca5 asdemonstrated by a colony formation assay fig jtogetherthese results demonstrated the roles ofcircsmarca5 in regulating the dna repair process inmcf7 cellstheevaluatecircsmarca5 overexpression enhances the cisplatinresponse in breast cancerto furthertherapeutic potential ofcircsmarca5 in breast cancer in vivo we establishedcircsmarca5 overexpression clones in mcf7 cells asshown in fig 8a the overexpression of circsmarca5efficiently enhanced the sensitivity of mcf7 xenograftsto concurrent cisplatin treatment fig 8a b the overexpression of circsmarca5 was confirmed by in situhybridization and qpcr analysis fig 8c along with decreased smarca5 protein levels and increased γh2axlevels fig 8d in addition qpcr analysis demonstratedthat circsmarca5 can be detected in the bloodsuggesting that circsmarca5 is a secretory moleculecollectively these data demonstrate that circsmarca5could serve as a potential therapeutic target to restoresensitivity to cisplatin therapy in breast cancerdiscussionprevious studies have indicated that circrnas have multiple functions in cancer development and progression[] in this study we identified multiple expressedcircrnas in breast cancer samples and observed averagehigher abundance of circrnas over their host genes inperipheral blood than tissues which might contribute tothe exploration of diagnostic biomarkerfor breastcancer we then identified that circsmarca5 is significantly decreased in breast cancer tissues using rnaseqmore importantly we define a critical role for circsmarca5 in the regulation of dna damage repaircapacity and the drug sensitivity of breast cancer cellsin vitro and in vivo through the negative regulation ofits parent gene smarca5 these findings are of highclinical relevance because chemotherapy with cisplatinand bleomycin remains the standard of care in breastcancer [] hence the restoration of circsmarca5levels provides an approach to overcome treatmentresistance in breast cancer patientssmarca5 also known as snf2h is a member of theswisnf chromatinremodeling complex during dnadamage repair processes smarca5 is recruited todna damage sites where it induces the ubiquitinationand phosphorylation of histone h2a which facilitateschromatin remodeling and dna damage repair [ ]in this study we show that circsmarca5 expressionresulted in the downregulation of smarca5 and theeffect of circsmarca5 overexpression on dna repaircapacity was reversed by concomitant smarca5 overexpression suggesting that the effect of circsmarca5on dna repair capacity is mediated through smarca5circrnas exert functions in various ways such as forming an rloop with dna to regulate splicing and transcriptional pausing for example circsepallata3regulates the splicing of its parent mrna through rloop formation in addition circrnas are a novelclass of cernas that sponge mirnas thus positivelyregulating gene expression [ ] additionally circrnas such as exonintron circrnas regulate geneexpression through specific rnarna interactions withu1 snrna furthermore circrnas also exertfunctions by binding to proteins and regulating theiractivities we identified one mechanism by whichcircsmarca5 regulates the drug sensitivity of breastcancer cells to cisplatin and bleomycin through thedownregulation of smarc5 circsmarca5 is recruitedto its parent gene locus leading to rloop formationtranscriptiontruncatedδsmarca5 protein upregulation and decreased smarca5 expression this regulatory mechanism has alsobeen verified in cervical cancer hela cells figure s14however our evidence demonstrates that circsmarca5has no significant effect on the proliferation migrationand apoptosis of breast cancer cells suggesting that thismolecule functions in a celltype and contextdependentmanner notablythatnonfunctionalterminationweprovideevidence 0cxu molecular cancer page of fig circsmarca5 can form an rloop with its parent gene a secondary structure prediction for circsmarca5 using the mfold program thesequence key shared by the minimum free energy structure and the thermodynamic ensemble structure is marked by red b thethiophosphorus nucleic acid analog ant complementary to key and its mutant antmut were synthesized in vitro c dotblot verifying theinteraction between circsmarca and ant or antmut d dripqpcr analysis on exon or exon sequences of smarca5 to detect theassociation of circsmarca5 in mcf7 cells overexpressing ant or antmut rnase htreated andor dripqpcr analysis of the exonsequence as a control indicates p e western blot analysis shows that transfection of ant into circsmarca5overexpressing cells canrestore smarca5 protein levels but antmut cannot f g dotblot analysis quantifying rloop strength between the smarca5 locus andcircsmarca5 or circsmarca5mut guanine converted to cytosine of the key sequence h dripqpcr in mcf7 cells transfected withcircsmarca5 or circsmarca5mut rnase htreated genomic dna and qpcr of exon1314 were treated as controls indicates p iwestern blot analysis shows that overexpression of circsmarca5 to mcf7 cells can decrease smarca5 protein levels but circsmarca5mutcannot 0cxu molecular cancer page of fig circsmarca5 decreases dna repair capacity a circsmarca5 increases sensitivity to cisplatin or bleomycin in mcf7 cells mcf7 cellsstably expressing control vector or plcdhcircsmarca5 were treated with cisplatin or bleomycin for h and cck8 was used to measure cellviability b relative colony formation units of mcf7 cells stably expressing control vector or plcdhcircsmarca5 treated with μm cisplatin or μgml bleomycin after hours the drugs were replaced by fresh medium the number of colonies was quantified c d e single cell gelelectrophoresis scge assay indicating that circsmarca5 overexpression inhibits cell recovery from dna damage mcf7 cells stably expressingcontrol vector or plcdhcircsmarca5 treated with μm cisplatin or μgml bleomycin after incubation for h the cells were recoveredwith fresh medium for or hours and then collected for scge analysis c immunofluorescence assay using an antiγh2ax antibody d andwestern blot assay with the indicated antibodies e f rtqpcr assay showing the relative levels of several key cell cycle genes in mcf7 cellsstably expressing control vector or plcdhcircsmarca5 treated with dmso or μm cisplatin for h and replaced with fresh medium for h indicates p g scge assay showing that the cotransfection of ant in circsmarca5overexpressing cells can restore the dna repaircapacity but the cotransfection of antmut cannot h scge assay showing that the overexpression of circsmarca5 in mcf7 cells can decreasedna repair capacity but the overexpression of circsmarca5mut cannot i smarca5 abrogates γh2ax levels induced by circsmarca5 mcf7cells were infecte | 0 |
" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [] an rln countof ¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1t3tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd students t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor cipreferenceage ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3rlnslymph node metastasislnr lodds ptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were à ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ¥ characteristicsunivariateormultivariateor cipreference age ¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ¥ glasa ¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3lymph node metastasisptnm stage ¥ iiilauren subtypeintestinaldiffusemixedunknown cireferencereferencereferencep bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ¥ deng proposed an optimal rln count of ¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncolamin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more personalized approach tocancer staging ca cancer j clin okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol tóth d bÃró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg 0c" | 0 |
Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A casecontrolstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction HardyWeinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 oddsratio OR confidence interval CI however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [] Graves ophthalmopathyGO is one common anspecific complication affecting of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO []Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acasecontrol study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbeccos modified Eagles medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at ¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturers protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3CMycNR3C1 pCMV3CMycFOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturers protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturers protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNAIP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM TrisHCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydIdC and glycerol] for min at ¦C in a final volume of μl FOXP3 or NR3C1nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive [] [] Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbSigniï¬cance of age were evaluated by t testP005P00010039a 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the HardyWeinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 OR CI which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG 0929bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with signiï¬canceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAG2495cGG b0154b2467bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with signiï¬canceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearsons correlation Spearmans correlation Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearsons correlation Spearmans correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3CE The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 BE Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GOatherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via Drug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE HardyWeinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration inhibitory factor NR3C1 nuclear receptorsubfamily group C member OR odds ratio PCR polymerase chain reaction qDNAIP quantitative DNA immunoprecipitation SNP singlenucleotide polymorphism T3 triiodothyronine T4 free thyroxine TRAb thyrotropin receptor antibody TSHthyroid stimulating hormoneReferences Smith TJ and Hegedus L Graves Disease N Engl J Med 101056NEJMra1510030 Brent GA Clinical practice Graves disease N Engl J Med 101056NEJMcp0801880 Ginsberg J Diagnosis and management of Graves disease CMAJ Canadian Med Assoc J J de lAssoc Med Canadienne The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20202072101042BSR20202072 McIver B and Morris JC The pathogenesis of Graves disease Endocrinol Metab Clin North Am 101016S0889852905702991 Bednarczuk T Gopinath B Ploski R and Wall JR Susceptibility genes in Graves ophthalmopathy searching for a 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"Silent lunch and tea break 7. Taking care of yourself - Sitting meditation ending in choiceless awareness - Exercise on taking care of yourself by examining how to improve balance in life - Meditation without CD - Yoga or walking meditation - Reflect on training - 3-min breathing space 8. The rest of your life - Bodyscan - Reflection on training - Further sources of information - Short sitting meditation - Maintaining practice Outcome measures Primary outcome measure Psychological distress The primary outcome measure is the total score on the HADS [39-41] which is developed to measure psychological distress in somatic patient populations. It consists of a 7-item anxiety (HADS-A) and 7-item depression (HADS-D) subscale. The HADS shows good psychometric properties in the general medical population including oncology patients [42]. Internal consistency as measured with Cronbachs ? varied from .84 to .90 [4042].Test-retest reliability was good as Pearsons r > .80 were obtained [4043]. Though the cut-off scores of the HADS vary among populations [44] in lung cancer patients they have found to be <8 versus ?8 on the HADS-A or HADS-D [45]. The HADS has been shown to be highly correlated with the Beck Depression Inventory [42]. It has previously been used in intervention studies of mindfulness and shown to be sensitive to change (e.g. [46]). Secondary outcome measures Quality of life (only for patients) The European anisation for Research and Treatment of Cancer (EORTC) Core Quality of Life Questionnaire (QLQ-C30) [47] is included along with the supplemental Lung Cancer questionnaire module (QLQ-LC13) [48]. The QLQ-C30 is designed to use in clinical trials on physical treatments for cancer patients. It incorporates five functional scales (physical role cognitive emotional social) three symptom scales (fatigue pain nausea and vomiting) a global health and quality of life scale and an array of single-item symptom measures. After revisions in the role functioning global health and physical functioning scale internal consistency of the subscales varied between .65 and .94 except for the cognitive functioning scale with ? varying from .56 to .63 [474950]. Test-retest reliability varied from .63 to .86 [51]. The lung cancer questionnaire module is designed to supplement the core questionnaire and comprises specific symptoms associated with lung cancer (coughing haemoptysis dyspnoea pain) and side-effects from conventional chemo- and radiotherapy (hair loss neuropathy sore mouth dysphagia). While the multi-item dyspnoea scale showed high internal consistency the pain subscale did not. When combined with the dyspnoea and pain items of the core questionnaire both the dyspnoea (? = .86) and pain (? = .71) subscale showed high internal consistency. Since the QLQ-C30 and QLQ-LC13 are mainly focused on physical symptoms we added the items Social Interaction and Alertness Behavior of the Sickness Impact Profile (SIP) [52]. Internal consistency was .94 and test-retest reliability was .92. The SIP correlated with self-assessed sickness and dysfunction [52]. Caregiver appraisal (only for partners) We use the 9-item Self-Perceived Pressure from Informal Care (SPPIC) [53] to assess the extent to which caregiving is experienced as burdensome. To also measure positive aspects of caregiving the 9-item subscale Care-Derived Self-Esteem of the Caregiver Reaction Assessment (CRA-SE) [54] is included. Internal consistency of the SPPIC was .79 and of the CRA-SE was .73. The SPPIC and CRA-SE were unrelated to each other [55]. Relationship quality To measure relationship satisfaction we included the 10-item Satisfaction subscale of the Investment Model Scale (IMS-S) [56]. The IMS-S starts with 5 items that measure concrete examplars of satisfaction to enhance the comprehensibility of the global items which are utilized to form the construct. Internal consistency varied from .79 to .95 and the IMS-S was related to the Dyadic Adjustment Scale. Also the Mutual Interpersonal Sensitivity scale (MIS) [57] is included to measure communication between partners about the cancer. It contains 18 items and is divided into two scales: open communication and avoiding negative thoughts about the cancer. Spirituality is measured with the Spiritual Attitude and Involvement List (SAIL) [58] and consists of 26 items divided into the subscales meaningfulness trust acceptance caring for others connectedness with nature transcendent experiences and spiritual activities. The internal consistency varied from .74 to .88 and test-retest reliability varied from .77 to .92. All subscales except for connectedness with nature were related with the Functional Assessment of Chronic Illness Therapy Spiritual Well-Being Scale. Costs (only for patients) The cost-effectiveness evaluation is carried out from a societal perspective considering direct as well as indirect health costs. Data on costs are collected prospectively using a diary in which participants register a) health care utilization: the type of care and its duration and b) cancer-related absence from work. Unit cost estimates are derived from the national manual for cost prices in the health care sector [59]. Costs of reduced ability to work are estimated using the friction costs method which results in a more realistic estimate than the human capital approach [60]. Treatment costs of MBSR are calculated using activity-based-costing methods thus measuring actual resources (time of therapist time of patients facilities) used. All unit cost prices are adjusted to 2013 prices. Unit cost estimates are combined with resource utilization data to obtain a net cost per patient over the entire follow-up period. Process measures Mindfulness skills are examined with the 39-item Five Facet Mindfulness Questionnaire (FFMQ) [6162]. The FFMQ is based on an exploratory factor analysis of five mindfulness measures which allowed items from different instruments to form factors providing an empirical integration of these independent attempts to operationalize mindfulness. This led to the following five subscales: observing describing acting with awareness non-judging of inner experience and non-reactivity to inner experience. Internal consistency varied from .72 to .93 among the different subscales. Most subscales were related to meditation experience Psychological Well-Being scales and psychological symptoms including the Brief Symptom Inventory [61]. FFMQ is sensitive to change in mindfulness-based interventions and is found to mediate the relationship between mindfulness practice and improvements in psychological symptoms (e.g. [63]). Self-compassion is assessed with the Self Compassion Scale (SCS) [6465] which has 26 items and is divided into six subscales: self-kindness versus self-judgment common humanity versus isolation and mindfulness versus over-identification. Internal consistency of the different subscales varied from .75 to .81 and test-retest reliability varied from .80 to .93. SCS correlated moderately with self-esteem measures including the Rosenberg Self-Esteem Scale. Furthermore whereas the self-esteem measures correlated significantly with the Narcissistic Personality Inventory the SCS was unrelated to narcissism [64]. SCS is sensitive to change through mindfulness-based interventions and is found to mediate MBCTs treatment effects [66]. To measure rumination we administered the extended version of the Ruminative Response Scale (RRS-EXT) [67] Raes and Hermans: The revised version of the Dutch Ruminative Response Scale unpublished instrument]. The RRS-EXT contains 26 items in which a more adaptive thinking style (i.e. reflection) is distinguished from a more maladaptive one (i.e. brooding). Internal consistency varied from .72 to .77 and test-retest reliability varied from .60 to .62 for the brooding and reflection subscales. The concept of rumination seems to be sensitive to change through mindfulness-based interventions and has been shown to mediate the effect of MBSR on depressive symptoms in oncology patients [68]. The psychological stress reaction is measured with the 15-item Impact of Event Scale (IES) [6970] which assesses two categories of responses: intrusive experiences and avoidance of thoughts and images associated with the event. Internal consistency varied from .65 to .92 [71] and test-retest reliability varied from .79 to .87 among the subscales [69]. IES correlated with anxiety and depression subscales of the General Health Questionaire. Adherence to MBSR is assessed during the entire study period with a calendar on which participants in the MBSR condition fill out on a daily basis whether they adhere to the mindfulness exercises: either formal practice (e.g. meditation exercise like the bodyscan) informal practice (e.g. activity with awareness) or no exercise. Adherence to MBSR has been shown to mediate the effects of MBCT on depressive symptoms [72]. Statistical analysis plan Sample size To determine the required sample size first the sample size was calculated that would be needed for a simple t-test and subsequently it was corrected for clustering repeated measurements and baseline. A two-sided t-test on the total HADS score [3940] (i.e. our primary outcome measure examining psychological distress (HADS-total) anxiety symptoms (HADS-A) and depressive symptoms (HADS-D)) would require 64 participants in each group to have 80% power to detect a medium-sized difference (effect size = 0.5) with alpha = 0.05. To correct for clustering we multiplied this sample size of 64 with the design factor (1 + (n ? 1) * ICC) where n denotes the cluster size and where ICC denotes the intra-cluster correlation. In our study the treatment groups will consist of 14 people of whom about 7 will be patients. With n = 7 and an estimated ICC = 0.01. [72] the correction factor equals 1.06. To correct for repeated measurements and the use of the baseline measurement as a covariate we multiplied the required sample size by the design factor 1+?/2??02 where ? denotes the correlation between the post-treatment HADS measurements and ?0 denotes the correlation between the baseline HADS with the post-treatment HADS measurements. With ? = 0.8 and ? = 0.5 as conservative estimates the second design factor equals 0.65. Consequently after correction for clustering and covariates we arrived at a required sample size of 0.65 * 1.06 * 64 = 44 patients per arm. So 88 patients with lung cancer would be required for the study. Based on our pilot study [van den Hurk Schellekens Molema Speckens and van der Drift in preparation] we expect a 20% drop-out rate. Therefore we intend to include 110 patients and 110 partners. Primary analyses The samples of lung cancer patients and partners will be analyzed separately. Baseline characteristics of the population will be compared between MBSR and control group to ensure that key variables were evenly distributed by randomization. First analyses will be based on the intention-to-treat approach. Next we will perform per-protocol analyses with the treatment-adherent sample (i.e. in the MBSR condition participants have to attend at least four of the eight MBSR sessions [73] and in the TAU condition participants do not attend a mindfulness-based programme). We will use linear mixed models to analyze all outcome variables (i.e. psychological distress quality of life (only for patient) caregiver appraisal (only for partner) relationship quality and spirituality) with treatment as fixed factor baseline measurement as covariate and a random intercept based on MBSR group. This procedure will use all observed data in our analyses. In addition Cohens d effect size [74] will be reported based on the difference between the group means on baseline and follow-up scores divided by the pooled standard deviation at baseline and follow-up. Secondary analyses Cost effectiveness The quality of life measures (i.e. QLQ-C30; QLQ-LC13) will be used to calculate Quality of Adjusted Life Years (QALYs) for each individual. Costs and effects (in terms of QALYs) will be combined in the incremental cost-effectiveness ratio (ICER). The ICER expresses cost-effectiveness in terms of incremental costs per QALY gained. To estimate confidence intervals for the mean of the ICER a non-parametric bootstrapping method will be used performing 1000 replications of the original data. In order to express the implications of the cost-effectiveness results more clearly a cost-acceptability curve will be constructed. In case of dominance a full cost analysis will be conducted to estimate the mean savings per patient per year. Mediation analyses To examine the possible underlying mechanisms of change in MBSR mediation analyses will be conducted. Only the data of the treatment-adherent sample will be included in these analyses. By means of a multiple mediation model suggested by Preacher and Hayes [75] we will test the mediating effect of mindfulness skills self-compassion rumination and adherence to MBSR on psychological distress quality of life (only in patients) caregiver appraisal (only in partners) relationship quality and spirituality. Discussion In the last ten years MBSR has not only proven to be a feasible and acceptable intervention in cancer patients [76] but it also seems to be effective in reducing psychological distress [30]. However the generalization of these results is limited because most participants were female patients with breast cancer. A large part of lung cancer patients already have advanced cancer at time of diagnosis and are confronted with a poor prognosis and low health status. Consequently they more often report psychological distress than patients with other diagnoses of cancer [89]. Hence it is not yet clear whether MBSR is a feasible acceptable and effective intervention in patients with lung cancer. Moreover little is known about the effectiveness of MBSR in partners of cancer patients [30] though they also often report psychological distress. Our pilot study of 19 lung cancer patients and 16 partners participating in an MBSR course provides preliminary evidence that MBSR is feasible and acceptable in this population (van den Hurk Schellekens Molema Speckens and van der Drift in preparation). The current trial will answer the question whether MBSR is effective in patients with lung cancer and their partners. We started enrolment of participants in February 2012. At the moment we think recruiting a sufficient number of patients and partners will be a challenge due to rapidly fluctuating health status and sudden changes in cancer treatment [77]. The main reasons for declining participation in patients is being too ill or that it is too much of a burden during chemo and/or radiotherapy. Furthermore no perceived need or motivation for the training is commonly mentioned. Among partners participation is highly depending on whether the patient is willing to participate. Although partners can take part separately partners who are interested do often not participate when the patients decline participation. Considering the difficulty of studying lung cancer patients and their partners [77] our trial will offer valuable information on whether MBSR as one of the few available psychosocial care programmes contributes to the alleviation of their psychological distress. Abbreviations MBSR: Mindfulness-based stress reduction; RCT: Randomized controlled trial; RUNMC: Radboud University Nijmegen Medical Centre; MBCT: Mindfulness-based cognitive therapy; MMSE: Mini mental state examination; DT: Distress thermometer; HADS: Hospital anxiety and depression scale; QLQ-C30: Quality of life cancer; QLQ-LC13: Quality of life lung cancer; SIP: Sickness impact profile; SPPIC: Self-perceived pressure from informal care; CRA-SE: Caregiver reaction assessment care-derived self-esteem; IMS-S: Investment model scale-satisfaction; MIS: Mutuality and interpersonal sensitivity; SAIL: Spiritual attitude and involvement list; FFMQ: Five facet mindfulness questionnaire; SCS: Self-compassion scale; RRS-EXT: Rumination response scale extended version; IES: Impact of event scale. Competing interests The authors declare that they have no competing interests. Authors contributions All authors contributed to the design of the study. AS MD and JP are the principal investigators of the study. MS drafted the paper which was modified and supplemented by all other authors. DH MS and MD are involved in recruiting participants while MS and DH take care of the logistics of the study and data collection. RD contributed specifically to the statistical analysis plan and WW contributed specifically to the design of the cost-effectiveness evaluation. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/14/3/prepub Acknowledgements This research is funded by Foundation Alpe dHuZes and the Dutch Cancer Society (Grant number KUN 20115077 awarded to Prof. dr. Anne E. M. Speckens Dr. Miep A. van der Drift and Prof. dr. Judith B. Prins). 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mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5 | 0 |
"researchWhat are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records dataHelen Strongman Rachael Williams2 Krishnan Bhaskaran1To cite Strongman a0H Williams a0R Bhaskaran a0K What are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records data BMJ 202010e037719 101136bmj 2020037719 º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received February Revised April Accepted July Authors or their employers Re use permitted under CC BY Published by BMJFor numbered affiliations see end of Correspondence toDr Helen Strongman helen strongman lshtm ac ukObjectives To describe the benefits and limitations of using individual and combinations of linked English electronic health data to identify incident cancersDesign and setting Our descriptive study uses linked English Clinical Practice Research Datalink primary care cancer registration hospitalisation and death registration dataParticipants and measures We implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and We calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers We described completeness of grade and stage information in the cancer registration data setResults gold standard cancers were identified Positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate Sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone For case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer When primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed Completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancersConclusions Ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority Strengths and limitations of this study º This is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in England to identify cases of the most common incident cancers º Using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º We described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º A key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataINTRODUCTIONThe Clinical Practice Research Datalink CPRD provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 Use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 In the field of cancer epidemiology CPRD primary care data linked to Hospital Episode Statistics Admitted Patient Care Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access data HES APC Office of National Statistics ONS mortality and National Cancer Registration and Analysis Service NCRAS cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment Use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for NCRAS data Research teams therefore commonly choose not to use all available linked data3 Cancer epidemiology studies can also be conducted using NCRAS and HES APC data provided by National Health Service NHS Digital and Public Health England PHE without linkage to CPRD primary care data4 This provides national coverage at the expense of the detailed health data that are available in primary care recordsValidation studies assessing concordance between CPRD GOLD HES APC and NCRAS data have estimated high positive predictive values PPVs for CPRD GOLD data and varying proportions of registered cancers that are not captured in CPRD GOLD and HES APC5 The most up to date analysis by Arhi et al included the five most common cancers and all papers focussed on concordance between CPRD GOLD only and NCRAS existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs National data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and Clinical Commissioning Groups10 We aim to describe and compare the benefits and limitations of using different combinations of linked CPRD primary care data HES APC ONS mortality and NCRAS cancer registration data for conducting cancer epidemiology studies Our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets We have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets We also describe the availability of stage grade and treatment variables over time in the cancer registration data for the CPRD linked cohort This reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesMETHODSStudy design and settingWe completed a concordance study using linked2 English CPRD GOLD HES APC ONS mortality and NCRAS data CPRD GOLD data were extracted from the January monthly release and the 13th update to CPRDs linked data The study period was from January to December with December matching the end of the NCRAS coverage periodThe CPRD GOLD database includes de identified records from participating general practices in the UK who use Vision software1 General practice staff can record cancer diagnoses using Read codes or in free text comments boxes though the latter are not collected by CPRD Diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care CPRD GOLD data are linked to HES APC ONS mortality and NCRAS through a trusted third party for English practices that have agreed to participate in the linkage programme2 HES APC data are collected by NHS Digital to co ordinate clinical care in England and calculate hospital payments12 Admissions for and related to cancer diagnoses are recorded using International Classification of Diseases version ICD10 codes National cancer registration data are collected by NCRAS which is part of PHE in accordance with the Cancer Outcomes and Services Data set13 which has been the national standard for reporting of cancer in England since January Data include ICD10 codes to identify the cancer site and more detailed information such as stage and grade ONS mortality data includes dates and causes of deaths registered in England recorded using ICD10 codesParticipants exposures and outcomesOur underlying study population included male and female patients registered in CPRD GOLD practices who were eligible for linkage to HES APC NCRAS and ONS mortality data and had at least days of follow up between January and December Start of follow up was defined as the latest of the current registration date within the practice and the CPRD estimated start of continuous data collection for the practice up to standard date End of follow up was determined as the date the patient left the practice ONS mortality date of death or practice last collection dateIdentification and classification of cancer codesWe used code lists to classify cancer records in each of CPRD GOLD HES APC and ONS mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https data Incompletely specified cancer codes could be mapped to cancer site eg ICD10 code C689 Malignant neoplasms of urinary an unspecified was considered consistent with both bladder and kidney cancer For NCRAS we accessed coded records for the most common cancers We included cancers recorded in the clinical or referral file for CPRD GOLD cancers recorded in any diagnosis field for HES APC and the underlying or Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Gold standard algorithm to identify incident site specific cancers using all data sources HES Hospital Episode Statistics NCRAS National Cancer Registration and Analysis Service ONS Office of National Statisticsmost immediate cancer cause of death in ONS mortality dataCancer case definitions based on individual sources and combinations of sourcesWe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources NCRAS alone NCRAS and HES APC all sources CPRD GOLD HES APC and ONS mortality CPRD GOLD alone HES APC alone Multiple malignant cancers recorded on the index date in CPRD GOLD or HES APC were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the NCRAS data if available and as multiple site cancer if not For each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upGold standard cancer case definitionWe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure Cancers recorded in NCRAS alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in HES APC alone or GOLD alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period Where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in NCRAS and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in NCRAS if there were more data sources in total with records for cancer at that site than data sources with contradictory recordsWe used NCRAS data to identify stage grade and treatment where available in the cancer registry only cohort Binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisStatistical analysisFor each cancer site and each individual or combined data source we combined our applied study definitions Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordWe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex We calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourcesWe used Kaplan Meier methods to describe mortality over time for cancers identified using each definition The ONS mortality death date was used for these analysesWe used the NCRAS only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timePatient public involvementPatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsRESULTSOf research quality patients in the CPRD GOLD January build were eligible for linkage to HES ONS mortality and NCRAS data in set were excluded due to unknown sex Of the remainder and had at least year of follow up between January and December and were included in the study population Using the gold standard algorithm incident cases of cancer were identified The number of patients identified with each cancer is presented in online supplementary appendix table Half n82 of these patients were male aged to aged to and aged or olderFigure presents PPVs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm When using NCRAS data alone to of cancers were confirmed by the algorithm for out of cancer sites the NCRAS only case definition gave the highest PPV Case definitions using data sources not including NCRAS generally had lower PPVs ranging from to for individual cancer sites For the four most common cancers breast lung colorectal and prostate PPVs were at least for all case definitions Minimal differences in PPVs were observed between age groups years and sexes online supplementary appendix figures Figure presents sensitivity values for each case definition Sensitivity was generally higher for the case definitions that included NCRAS data ranging from to for individual cancer sites except bladder cancer identified using NCRAS data alone and ¥ for the four most common cancers breast lung colorectal and prostate Sensitivity was also generally high for definitions using a combination of CPRD GOLD HES APC and ONS mortality data ranging from to ¥ for the four most common cancers Sensitivity was lower for case definitions that used CPRD GOLD alone range to for individual cancer sites or HES APC alone range to Sensitivity values for CPRD GOLD alone and HES APC alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures Post hoc analysis suggested that the low sensitivity of CPRD GOLD only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary an cancer codes n1108 in CPRD GOLD rather than contradictory information about the first cancer record n625 These incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 Bladder cancers that were not recorded in NCRAS data n3445 were commonly recorded in both HES APC and CPRD GOLD n2228 or in HES APC only with a subsequent unspecified or bladder cancer record in HES APC within months n995 Table describes the number of days median IQR and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used Case definitions using NCRAS alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using CPRD GOLD alone and HES APC aloneFigure describes mortality over time following incident cancer diagnoses ascertained from each case definition Minimal differences in mortality were observed between cancers identified from different case definitions Where variability was observed cancers identified using CPRD GOLD only had the lowest mortality rates eg kidney cancer and cancers identified using HES APC only or NCRAS only had higher mortality rates eg prostate cancer and bladder cancer respectivelyFigure describes completeness of grade and stage for cancers identified using NCRAS only Recording of grade was highly variable between cancers with gradual increases in completeness over time Completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in Post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure Online supplementary appendix figure describes recording of treatment modalities identified using NCRAS Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Positive Predictive Value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONSOffice of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0celitnecrep hthtRQI inadeMelitnecrepRQI inadeM hthtelitnecrep htht inadeMRQIelitnecrep hthtCPASEH DLOGDRPC ytil atromSNOdna CPASEH DLOGDRPC CPASEHdnaSARCN secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emTi l ebaT access recnaC lanoitaN SARCN atad eraC tneitaPdetti mdA scitsitatS edospEi latipsoH CPASEH knil ataDhcraeseR ecitcarP lacniilC DRPC metsys suovren lartnec SNCl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iiA setis recnac nommoc tsom ruoFDCiI nosrev sesaesDi fonoitacfissaCliscitsitatS lanoitaN rof ecfifO SNOi atadnoitartsgerrecnac ecvreS ssyanAdna liinoitartsgeRi lanoitanretnI eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaCi snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmuN ot ot ot ot ot Cl amoeym epitluMl ot Ci ameakueL ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtSARCN inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot C ytivac larOC laegahposeOC hcamotS CC latcerooClrecnaC amonaeml tnangilaMC saercnaPC gnuLC suretUC etatsorPC seiravOC yendKiC tsaerBCi xvreCCC SNCnarBiC reddaBlC amohpmyl s'inkgdo HnoNC idoryhTC revLiStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Mortality following first ever record of cancer in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphoma ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Completeness of grade and stage for cancers identified using NCRAS data only Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites Grading information is not applicable to brainCNS sarcoma or haematological cancers and not required by in the national data standard COSD for prostate cancer Core staging is not applicable to haematological and gynaecological cancers Other types of staging are recommended by COSD CNS central nervous system COSD Cancer Outcomes and Services Data set NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphomaStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access only Missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordedDISCUSSIONStatement of principal findingsWe investigated the use of different sources of electronic health record data to identify incident cancers For all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers Use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers Combining CPRD GOLD HES APC and ONS mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers Sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone Use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date Finally while we observed minimal changes in PPVs and sensitivities between and completeness of NCRAS cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 Completeness of cancer treatment recording was difficult to assess due to the absence of a missing categoryStrengths and weaknesses of the studyThe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from CPRD to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies We have also assessed the value of using NCRAS cancer registration data to measure stage grade and cancer treatment modalitiesA limitation of the study is that our gold standard algorithm is not validated We feel that we were justified in pre weighting NCRAS data as more reliable that other data sources as NCRAS is a highly validated data set that matches merges and quality checks data from multiple sources4 We did not consider NCRAS to be the outright gold standard as it is plausible that NCRAS does not identify all tumours diagnosed and treated in primary and secondary care For most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in HES APC CPRD GOLD or ONS mortality data but not in NCRAS These tumours may have been diagnosed and coded as invasive in primary or secondary care but not by NCRAS been incorrectly coded in HES APC CPRD GOLD or ONS mortality data not have been notified to NCRAS eg tumours treated in private hospitals or be the result of linkage errors between the data sets The proportion of cancers identified in HES APC but not in NCRAS is particularly high for bladder cancer This is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in NCRAS which are greatest for bladder cancer4 This explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in NCRAS compared with other data sources To identify incident cancers we required months of research quality follow up in CPRD GOLD prior to inclusion in the study Previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 The identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as NCRAS data collection started in HES APC in and ONS mortality data in and by the inclusion of all diagnostic codes in HES APC assuming that the first ever primary or secondary record identified incident cancer Reassuringly PPVs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases Requiring internal confirmation within months for cancers recorded in CPRD GOLD alone in our GOLD standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up Our data cut only included NCRAS data for the top cancers earlier cancers at other sites will have been missed in this studyIt is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesStrengths and weaknesses in relation to other studies discussing important differences in resultsThe most up to date study describing concordance between linked CPRD GOLD HES APC and NCRAS data sets demonstrated that to of the five most common cancers recorded in CPRD GOLD are not confirmed in either HES APC or cancer registration data and to of registered cancers are not recorded in CPRD GOLD8 For cancers recorded in both sources the diagnosis date was a median of to days later in CPRD GOLD than in the cancer registration data Using CPRD GOLD alone to identify these Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis Use of HES APC only identified a higher proportion of patients with the correct diagnosis date than CPRD GOLD but over represented older patients and those diagnosed through the emergency route The majority of registered cancers were picked up using both CPRD GOLD and HES APC ranging from for lung cancer to for breast cancer Previous research demonstrated similar results with substantial differences between cancer types5 Additionally a study using data from to found that using HES data in addition to NCRAS data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16Our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancersWe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards Levels of data completeness of staging information in the CPRD extract in were similar to those reported by the United Kingdom and Ireland Association of Cancer Registries UKAICR9Meaning of the study possible explanations and implications for clinicians and policymakersUse of NCRAS cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately Case definitions based on a combination of CPRD GOLD HES APC and ONS mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersThese findings | 2 |
neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing antiammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these ï¬nd ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the beneï¬t of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as insilico drug re purposing in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to ï¬nd out a new se ries of nep inhibitors to the best of our knowledge this is the ï¬rst study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of Ëa prior to docking and simulation studies the biological unit of protein was prepared using protein preparation wizard in schrodinger suite during the process of protein preparation the protein was subjected to import and reï¬ne review and modify and minimize processes in protein preparation wizard missing side chains and residues were ï¬lled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond Ëa were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force ï¬eld and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workï¬ow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force ï¬eld in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding aï¬nities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer ï¬le of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force ï¬eld to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme proï¬le the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coeï¬cient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of ï¬ve were calculated induced ï¬t docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedï¬t docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifdsp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which reï¬nement of all residues within Ëa of the ligands pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedï¬t protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula prime_energy glide score ifd score glide_ecoul molecular dynamics md simulation the ï¬exibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede ï¬ned spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol Ëa balanced at k tem perature and bar pressure via npt ensemble in the ï¬nal step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme proï¬le further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workï¬ow was used and the cubic box of speciï¬c dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the ï¬nal torsional reï¬nement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were ï¬ltered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identiï¬ed for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this ï¬nding indicates that the selected drugs may act as nep inhibitors induced ï¬t docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced ï¬t docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the ï¬exible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications ï¬rst it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd conï¬rmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme proï¬le was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of ï¬ve given in table all the selected drugs obey the lip inski rule of ï¬ve molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited ï¬exibility which is insuï¬cient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and lig ï¬t prot for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be Ëa and Ëa respectively the rmsd values were found to be in the acceptable range Ëa but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are Ëa and Ëa for protein and ligand respectively for complex the rmsd values were found to be Ëa for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of Ëa for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signiï¬ 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of ï¬ve sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is antiammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding aï¬nity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are ï¬rst it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drugs activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identiï¬ed based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0 | 0 |
Paired box protein8 PAX8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors This study aimed at investigating the immunohistochemical expression of PAX8 among Sudanese females diagnosed with cervical endometrial and ovarian cancers between December and May by studying their Formalinfixed paraffin embedded blocksResults Sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range Cervix was the most common cancer site in patients Regarding cancer stage there was and of the study population had stage 3B and 2B respectively The histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma SCC as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively PAX8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated SCC All patients diagnosed with well differentiated SCC and metastatic adenocarcinoma showed no expression of PAX8 A statistically significant was seen for PAX8 expression and the different histopathological diagnosis P value Keywords Female reproductive cancer Paired box protein8 Immunohistochemical expressionIntroductionPaired box protein8 PAX8 is a member of the family paired box proteins PAXs [ ] PAX8 consists of amino acids with a molecular weight of approximately kilo Dalton and its molecular properties are located on chromosome 2q13 [] PAX8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] During the embryonic period PAX8 also plays a significant role Correspondence nouh_saadoutlookcom Alfarrabi College for Science and Technology Khartoum SudanFull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the M¼llerian ducts [] In a previous experiment the deletion of the PAX8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal ing Also resulted in poor development of the myometrial tissue [] Several studies have described the immunohistochemical utility of PAX8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]In a healthy female reproductive tract PAX8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] PAX8 was found to be expressed among endometrioid carcinomas transitional The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cAli a0et a0al BMC Res Notes Page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] Whereas for the ovarian carcinomas PAX8 was under expressed [] Considering that few studies have investigated the immunohistochemical expression of PAX8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from Sudan yet [ ] This study aimed at investigating the immunohistochemical expression of PAX8 among Sudanese females diagnosed with cervical endometrial and ovarian carcinomasMain textMaterials and a0methodsStudy design and a0population characteristicsThis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from December till May in Khartoum State Sudan We retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas The retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis The participants demographic data was collected including age place of residence The clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedSections Preparation for a0Immunohistochemistry StainingTwo sections were cut using Rotary microtome Leica Germany from each histopathological block Then one slide was stained by hematoxylin and eosin staining technique The other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry To retrieve PAX8 tissues antigen we treated the sections with citrate buffer at ° a0C for a0min in a waterbath Then the tissue sections were rinsed first in distilled water and later with Tris buffer saline TBS This was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity The slides were then placed in a humid chamber Then the slides were drained and rinsed in two successive changes of Tris buffer wash buffer for a0 min each Nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min Then the remaining solution was drained from the slides The sections were then incubated in the primary antibody PAX8 antiPAX8 rabbit antihuman monoclonal antibody ab189249 Abcam United Kingdom at room temperature in the humid chamber according to the manufacture instructionsObserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the PAX8 For the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline PBSResults interpretationsFor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories Negative No staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker The slides were interpreted and validated by two expert pathologists blindly of each other results Photomicrographs were taken using Olympus SP350 camera Olympus Imaging America Inc USAStatistical analysisThe statistical analysis of the results was done using IBM SPSS Statistics vs The ChiSquared test was performed to compare the frequencies of categorical variables Statistical significance level was defined as p value at confidence intervalResultsCharacteristics of a0the a0study participantsThe study included patients diagnosed with female genital tract cancer Patients aged ± a0years range a0years Patients were grouped into age groups Those aged a0 years constituted half of the study participants The remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively According to patients place of residence patients were originating from the four regions of Sudan Most of the patients were from western part of Sudan followed by from the central part of SudanRegarding the site of cancer the cervix was the most commonly involved patients There were and endometrial and ovarian cancer respectively Based on the International Federation of Gynecology and Obstetrics FIGO cancer grading the majority of the study population was diagnosed with stage 3B and 2B cancer and of the patients respectively The were and stage 4B 3A 2A 1B and 4A respectively 0cAli a0et a0al BMC Res Notes Page of No statistically significant association between FIGO staging and age group was found P value Histologically there were squamous cell carcinoma SCC all of which were cervical cancers and adenocarcinoma SCC and adenocarcinoma were further classified into poorly differentiated SCC moderately differentiated SCC and well differentiated SCC endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomaBased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type FIGO staging and cancer histopathological type Table a0Immunohistochemical Expression of a0PAXThe immunohistochemical expression of PAX8 was shown as a yellowishbrown or brown staining of the nucleus Fig a0 Based on site of cancer all endometrium carcinoma showed positive expression of PAX8 with P value There were only patients who had positive expression of PAX8 including adenocarcinoma and SCC A statistically significant difference was noted for the PAX8 staining and cancer type with P value The analysis of PAX8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated SCC and metastatic adenocarcinoma had negative results for the PAX8 expression While of the endometrium adenocarcinoma were found positive for the PAX8 expression A statistically significan was t seen for PAX8 expression and the different histopathological diagnosis P value Table a0Table Classification of a0Participants demographic and a0clinical diagnosis based on a0age groupAge group no Total no P value a0years a0years a0years a0yearsResidence of patient Central Sudan East Sudan West Sudan North SudanSite of cancer Cervix Endometrium OvaryCancer histological type SCC AdenocarcinomaFIGO staging Stage Stage 2A Stage 2B Stage 3A Stage 3B Stage 4A Stage 4BHistopathological cancer grades Well differentiated SCC Poorly differentiated SCC Moderately differentiated SCC Endometrium adenocarcinoma Endocervical adenocarcinoma Metastatic adenocarcinoma Ovarian mucinous cyst adenocarcinomaSCC Squamous Cell Carcinoma 0cAli a0et a0al BMC Res Notes Page of Fig Immunohistochemical expression of PAX8 among the different histopathological cancer types and grades The immunohistochemical expression of PAX8 is shown as a yellowishbrown or brown staining of the nucleus a Well differentiated SCC negative b Metastatic adenocarcinoma negative c Poorly differentiated SCC positive d Moderately differentiated SCC positive e Endometrium adenocarcinoma positive f Ovarian mucinous cystadenocarcinoma positive g Endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cAli a0et a0al BMC Res Notes Page of Table Association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0PAX8PAX results no Total no P valuePositiveNegativeCancer histological type SCC AdenocarcinomaCancer site Cervix Endometrium OvaryFIGO staging Stage Stage 2A Stage 2B Stage 3A Stage 3B Stage 4A Stage 4BCancer histopathological grading Well differentiated SCC Poorly differentiated SCC Moderately differentiated SCC Endometrium adenocarcinoma Endocervical adenocarcinoma Metastatic adenocarcinoma Ovarian mucinous cyst adenocarcinomaSCC Squamous Cell Carcinoma DiscussionPrevious studies on the immunohistochemical expression of PAX8 in the normal female reproductive tract showed that PAX8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] This study investigated the immunohistochemical expression of PAX8 in Sudanese patients who were diagnosed with female reproductive tract cancers Patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer However previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]Regarding the place of residence the majority of patients coming from western Sudan This result is in contrary with a previous study in Sudan conducted by Saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern Sudan were higher compared to the other regions in Sudan [] Nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion Therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresThe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer Similar results were seen previously among Sudanese females [] Also the high frequency of stages 3B and 2B compared to the other stages were comparable to previous study conducted in Sudan [] This similarity underscores a delayed response among Sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young Sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentRegarding the classification based on the histopathological diagnosis most of the female diagnosed with SCC This result was also similar to previous study investigated the prevalence of the different gynecologic cancer in Sudan [] However the expression of PAX8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cAli a0et a0al BMC Res Notes Page of to the site of cancer development While agrees with another study where PAX8 was expressed only in patient []Interestingly a high frequency of PAX8 expression was noted among females diagnosed with endometrium cancer compared to SCC this finding is in contrary with a previous report where PAX8 was expressed among only of the studied samples [] Also the result was strongly in accordance with other studies [ ] Besides that the lack of PAX8 expression among those who were diagnosed with well differentiated SCC and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]ConclusionAlthough PAX8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated SCC and metastatic adenocarcinoma PAX8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractLimitations¢ The small sample size investigated in this study reduced the ability of using the expression of PAX8 as a diagnostic marker Therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemAcknowledgementsThe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyAuthors contributionsETA NSM and EES provided conceptual framework for the study guidance for interpretation of the data and performed data analysis ETA EES IRS LAH and AMM performed laboratory work NSM EES MSM AAY and AA performed the statistical analysis NSM MSM EES and AA participated in the manuscript preparation revision and coordination All authors read and approved the final manuscriptFundingNot ApplicableAvailability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Research Ethics Committee of the Faculty of Medical Laboratory Sciences University of Khartoum Sudan Ethical Approval No FMLSREC002042 All participant approved to participate by signing an informed consentConsent for publicationNot ApplicableCompeting interestsNo competing interests to discloseAuthor details Department of Histopathology and Cytology Faculty of Medical Laboratory Sciences University of Khartoum Khartoum Sudan Department of Histopathology and Cytology Faculty of Medical Laboratory Sciences National University Khartoum Sudan Alfarrabi College for Science and Technology Khartoum Sudan Faculty of Medicine Sinnar University Sennar Sudan Molecular Biology Department Faculty of Medical Laboratory Sciences Nile University Khartoum Sudan Faculty of Dentistry Ibn Sina University Khartoum Sudan Department of Neurology Mayo Clinic Jacksonville FL USA Department of Radiology Mayo Clinic Jacksonville FL USA Institute of Endemic Diseases University of Khartoum Khartoum Sudan Mycetoma Research Center University of Khartoum Khartoum Sudan Faculty of Medicine Nile University Khartoum Sudan Received July Accepted August References Gruss P Walther C Pax in development Cell Mansouri A Hallonet M Gruss P Pax genes and their roles in cell differentiation and development Curr Opin Cell Biol Macchia PE Lapi P Krude H Pirro MT Missero C Chiovato L Souabni A Baserga M Tassi V Pinchera A PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis Nat Genet Vilain C Rydlewski C Duprez L Heinrichs C Abramowicz M Malvaux P Renneboog Bt Parma J Costagliola S Vassart G Autosomal dominant transmission of congenital thyroid hypoplasia due 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ÎNp63 in squamous cell and urothelial carcinoma Am J Clin Pathol Yemelyanova A Gown AM Holmes BJ Ronnett BM Vang R PAX8 expression in uterine adenocarcinomas and mesonephric proliferations Int J Gynecol Pathol Liang L Zheng W Liu J Liang SX Assessment of the utility of PAX8 immunohistochemical stain in diagnosing endocervical glandular lesions Arch Pathol Lab Med Wong S Hong W Hui P Buza NJIJoGP Comprehensive analysis of PAX8 expression in epithelial malignancies of the uterine cervix Int J Gynecol Pathol De Andrade DAP Da Silva VD de Macedo MG De Lima MA de Andrade VM Andrade CEMC Schmidt RL Reis RM Dos Reis R Squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer PLoS ONE 20191410e0220086Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid 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Rheumatoid arthritis RA is a systemic chronic inflammatory disease that affects synovial joints and has various extraarticular manifestations including atherosclerotic cardiovascular disease CVD Patients with RA experience a higher risk of CVD leading to increased morbidity and mortality Inflammation is a common phenomenon in RA and CVD The pathophysiological association between these diseases is still not clear and thus the risk assessment and detection of CVD in such patients is of clinical importance Recently artificial intelligence AI has gained prominence in advancing healthcare and therefore may further help to investigate the RACVD association There are three aims of this review to summarize the three pathophysiological pathways that link RA to CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators useful for RA patients and to understand the role of artificial intelligence in CVD risk assessment in RA patients Our search strategy involves extensively searches in PubMed and Web of Science databases using search terms associated with CVD risk assessment in RA patients A total of peerreviewed s were screened for this review We conclude that a two of the three pathways directly affect the atherosclerotic process leading to heart injury b carotid ultrasound imagebased calculators have shown superior performance compared with conventional calculators and c AIbased technologies in CVD risk assessment in RA patients are aggressively being adapted for routine practice of RA patientsKeywords Arthritis a0· Rheumatoid a0· Atherosclerosis a0· Cardiovascular disease a0· Inflammation a0· Carotid artery diseases a0· Carotid intimamedia thickness a0· Risk assessmentIntroductionRheumatoid arthritis RA is a chronic inflammatory disease that not affects only synovial joints but also has several extraarticular involvements including those related to the skin eyes heart lungs kidneys and other ans [ ] It affects of the global population with a higher prevalence in females when compared with males [ ] Cardiovascular disease CVD is a common manifestation in RA patients with a two to threefold higher risk of cardiovascular events and mortality compared with a normal population [] However this increased risk is not entirely jasjitsuriatheropointcom Jasjit S Suri Extended author information available on the last page of the explained by conventional risk factors [] Current statistically derived CVD risk calculators use conventional risk factors alone [] are not suitable for RA patients and they either underestimate or overestimate the risk [] This may be because of the paradoxical behavior of some of the conventional risk factors such as body mass index lowdensity lipoprotein highdensity lipoprotein and total cholesterol in RA [ ] Despite this lack of clarity the guidelines by the European League Against Rheumatism EULAR recommend aggressive control of these conventional risk factors [ ] Recent attempts were made to improve the CVD risk assessment in the RA population including the development of RAspecific risk factors in the CVD risk calculators [] However such calculators could not provide adequate improvement in risk Vol01234567891 0c Rheumatology Internationalprediction and reportedly still underestimated or overestimated CVD risk in RA patients [ ]To provide a better CVD risk assessment in RA a pathophysiological association between these diseases should be understood as this would help in refining CVD risk predictors in RA patients [] Atherosclerosis a common phenomenon in RA [ ] can be adequately monitored using imaging modalities such as magnetic resonance imaging [] computed tomography [] optical coherence tomography [] and ultrasound [] Each of these imaging modalities offers unique information about morphological variations in atherosclerotic plaque Ultrasound imaging specifically in carotid arteries is a comparatively lowcost noninvasive radiationfree and easytouse imaging modality that is widely adopted in preventive cardiovascular and clinical vascular practices [ ] The imagebased phenotypes of carotid ultrasound such as carotid intimamedia thickness cIMT and carotid plaque are considered surrogate markers of coronary artery disease and have been used for preventive CVD risk assessments in several studies [] These imagebased phenotypes indicate the morphological variations in the atherosclerotic plaque and are associated with the inflammatory markers of RA [] Patients with RA have elevated cIMT and have more plaque area PA when compared with nonRA patients [] Thus the inclusion of these imagebased phenotypes in risk prediction models may improve the CVD risk assessments of RA patients Recent studies have combined the effect of these imagebased phenotypes with conventional risk factors including proinflammatory markers like erythrocyte sedimentation rate ESR to perform CVD risk assessment [] Such integrated risk calculators have demonstrated better CVD risk stratification when compared to traditional CVD risk calculators in nonRA patients [ ]Besides these statistically derived CVD risk calculators artificial intelligence AIbased techniques are also penetrating several medical imaging and risk assessment applications [] AIbased algorithms such as machine learning ML methods provide a better CVD risk assessment when compared with statistically derived conventional risk calculators [ ] So far AI algorithms have been used for CVD risk assessment in the nonRA population and their potential still needs to be evaluated in RA cohorts However AI is well adapted for RA screening and diagnosis [] This review provides an insight into how the AIbased algorithms can be used for CVD risk assessment in RA patients There are three aims of this review to summarize the pathophysiological pathways that link RA with CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators for RA patients and to provide an understanding of the role of artificial intelligence in CVD risk assessment in RA patientsSearch strategyFigure a0 shows a flow diagram indicating the search strategy for this narrative review To write a comprehensive narrative review it is essential to select at least two credible databases that provide highquality peerreviewed s [] This review is the outcome of several searches in the PubMed and Web of Science databases using keywords such as cardiovascular diseases AND risk assessment AND rheumatoid arthritis carotid atherosclerosis AND rheumatoid arthritis noninvasive imaging AND rheumatoid arthritis carotid ultrasound AND rheumatoid arthritis carotid intimamedia thickness OR carotid plaque AND inflammatory markers carotid atherosclerosis AND erythrocyte sedimentation rate OR C reactive protein machine learning AND rheumatoid arthritis and machine learning AND cardiovascular risk assessment AND rheumatoid arthritis The availability of all these keywords in the and the full text was investigated to select the relevant s Peerreviewed s published in the last a0years were then given priority Citations from the published s were also shortlisted for this review All these s were subsequently filtered by the expert coauthors to select only those that met the objectives of this review leading to sPathophysiology of a0RA leading to a0CVDThe pathophysiological association between RA and CVD can be explained in two stages the role of traditional risk factors and direct vascular damage Inflammation plays a pivotal role in both of these stages []The role of a0traditional risk factors in a0the a0pathophysiology of a0RAdriven atherosclerotic CVDThe righthand panel of Fig a0 explains the pathophysiological association between RA and CVD via four pathways [IaId] governed by traditional risk factors such as hypertension proatherogenic dyslipidemia insulin resistance and obesity Patients with RA are generally found with proinflammatory cytokines such as interleukin IL IL6 and tumor necrosis factor α TNFα [] These proinflammatory cytokines are found in the synovium which triggers a systemic inflammatory response and may result in damage to the vascular endothelial cells [] Nitric oxide NO and cyclooxygenase1 are two essential components required 0cRheumatology International Fig Flow diagram for the search strategyto maintain the healthy endothelium which is inhibited by TNFα and IL6 thereby resulting in endothelial cell damage [ ] Inhibiting endothelial NO leads to arterial stiffness [] and is further associated with an increase in peripheral vascular resistance PVR [] thus leading to hypertension in RA patients Additionally several medications used to treat RA such as diseasemodifying antirheumatic drugs DMARDs leflunomide and cyclosporine glucocorticoids nonsteroidal antiinflammatory drugs NSAIDs and cyclooxygenase II inhibitors Cox IBs may also be involved in the development of hypertension in RA patients [ ]Another pathophysiological link between RA and CVD is proatherogenic dyslipidemia [] Nearly of RA patients have proatherogenic dyslipidemia [] In nonRA patients increased CVD risk is associated with elevated levels of lowdensity cholesterol LDLc total cholesterol and reduced highdensity lipoprotein cholesterol HDLc However in RA patients low levels of total cholesterol TC low levels of LDLc and suppressed levels of HDLc have been reported This condition is known as the lipid paradox [] Highly suppressed HDL levels in RA patients are proatherogenic [] Furthermore RA patients show high atherogenic index levels despite low lipid levels The atherogenic index is calculated as a ratio of TC HDLc and it may vary according to their levels [] Apolipoprotein B Apo B is a major apolipoprotein in LDL and several studies have indicated an increase in the ratio of Apo B Apo A in RA patients [] A combination of low TC LDLc and suppressed HDLc levels with a high atherogenic index and a high ApoBApoA ratio behaves as proatherogenic dyslipidemia [ ] Longstanding proatherogenic dyslipidemia causes atherosclerosis and eventually CVDRheumatoid cachexia is another important RAspecific characteristic that increases CVD risk [] It is characterized by significantly increased adiposity and reduced muscle mass while one maintains their bodyweight [] The pathophysiology [shown in pathwayI c] behind R cachexia can be explained in two ways It is characterized by the reduction of muscle mass that is largely due to increased inflammatory cytokines particularly TNFα by activating the transcriptional nuclear factorkappa B cells NFkB pathway and promoting the ubiquitin pathway which causes catabolismproteolysis muscle protein breakdown [ ] Central obesity or abdominal obesity is present in of women and of men This causes visceral adiposity in RA which has an additional adverse impact on CVD [] On the other hand increased adiposity also induces the production of inflammatory cytokines in RA which further worsens this 0c Rheumatology InternationalFig Pathophysiological association between rheumatoid arthritis and cardiovascular disease IL1 interleukin IL6 interleukin TNFα tumor necrosis factor α EC endothelial cells SMC smooth muscle cells MCP1 monocyte chemoattractant protein MCSF macrophage colonystimulating factor VCAM vascular cell adhesion molecule ICAM intercellular adhesion molecule NSAIDs nonsteroidal antiinflammatory drugs CoxIBs cyclooxygenase inhibitors HTN hypertension PVR peripheral vascular resistance TC total cholesterol HDL highdensity lipoprotein LDL lowdensity lipoprotein APOB apolipoprotein B APOA apolipoprotein A NFkB nuclear factorkappa B cellsscenario [] This syndrome may be explained in the triad of increased adiposity reduced muscle mass and low body mass index BMIEpidemiological studies have suggested a strong association between insulin resistance IR metabolic syndrome and RA [ ] [shown in pathwayI d in the dark greendotted box] Inflammation plays a crucial role in these three conditions [] In patients with RA IR serves as an independent prognostic risk factor that signifies the presence of subclinical atherosclerosis it is determined by carotid intimal thickness cIMT and is measured by carotid ultrasonography [] Longstanding inflammation due to RA promotes oxidative stress endothelial dysfunction and atherosclerosis in this population []Progression of a0atherosclerosis and a0direct vessel damage in a0RAIn RA the activation of Tcells and mast cells increases the production of proinflammatory cytokines such as IL1 IL6 and TNFα These proinflammatory cytokines stimulate endothelial cells ECs and smooth muscle cells SMCs in subendothelium [] by expressing cell adhesion molecules such as vascular cell adhesion molecule VCAM and the intercellular adhesion molecule ICAM [] and by producing chemokines including monocyte chemoattractant protein MCP and macrophage colonystimulating factor MCSF The activation of endothelial cells allows the migration of LDLc into the subendothelial layer where 0cRheumatology International it becomes oxidized and triggers the inflammatory response for the recruitment of immune cells such as T lymphocytes and monocytes in the intimal layer Once they enter the intimal layer monocytes are transformed into macrophages and they then take up the oxidized LDLc to become foam cells The completion of this complex process then leads to the formation of atherosclerotic plaque Macrophages also trigger the migration of smooth muscle cells from tunica media to tunica intima and initiate their proliferation The SMCs form a thin fibrous cap to prevent the encroachment of atherosclerotic plaque towards the lumen However over time proinflammatory cytokines enzymes and free radicals cause fibrous cap erosion and make the plaque vulnerable for rupture The amplification of the inflammatory response results in the acceleration of plaque formation eventually leading to plaque rupture and thrombotic events which damage the blood vessels Pathway II of Fig a0 represents this processCurrent conventional CVD risk prediction models for a0RAOver the last decade several CVD risk assessment calculators have been developed very few of which are recommended by the cardiovascular risk management guidelines [ ] Some standard cardiovascular risk prediction algorithms are the Framingham risk score FRS [] Systematic Coronary Risk Evaluation SCORE [] American College of CardiologyAmerican Heart Association ACCAHA risk score [] World Health anization WHO risk charts [] and Reynoldss risk score RRS [] These risk calculators use traditional risk factors such as patient demographics age gender ethnicity blood biomarkers lowdensity lipoprotein cholesterol highdensity lipoprotein cholesterol and total cholesterol behavioral markers smoking and alcohol consumption and physiological markers height weight body mass index All these risk calculators were initially developed for nonRA populations therefore when used in RA cohorts CVD risk is substantially underestimated [] The use of traditional risk factors alone while not considering RAspecific inflammatory markers could be another reason for such underestimation However RRS included an RAspecific inflammatory marker called high sensitivity Creactive protein hsCRP [] for CVD risk prediction but did not report any significant improvement in the CVD risk assessment [] Rajagopalan et a0al [] also reported a small improvement in area under the curve in females and in males when C reactive protein CRP or erythrocyte sedimentation rate ESR was added to the FRSOver the past few years several efforts have been made to improve the cardiovascular risk assessment in RA patients The EULAR guidelines recommended the use of a modified SCORE mSCORE in RA patients positive with rheumatoid factor RF or anticitrullinated protein antibodies ACPA and RA duration of more than a0years [ ] Cox et a0al [ ] developed the QRISK2 and QRISK3 algorithms which use the presence of RA as a CVD risk predictor hazard ratio confidence interval Solomon et a0al [] also developed an RAspecific CVD risk calculator called expanded risk score or ERS by including RAspecific biomarkers [such as disease activity disease duration a modified health assessment questionnaire HAQ disability index and daily prednisone use] with the traditional biomarkers used in the Coxbased model The authors reported an improvement of in cindex when validating the risk score on the reserved dataset Recently Curtis et a0al [] also proposed a CVD risk prediction tool for RA patients by combining conventional and RAspecific risk factors The authors predicted the risk of composite CVD events such as MI stroke and death during the followup period of a0years The area under the curve AUC for cardiovascular risk stratification for this model was All these RAspecific CVD risk scores reported a better risk assessment on the proprietary databases Still when compared with other risk calculators in different RA cohorts these calculators have demonstrated mixed results [] Crowson et a0al [] reported an underestimation of CVD risk by FRS and RRS in RA patients The observed risk was twice the predicted risk Furthermore the authors did not report any improvement in cardiovascular risk prediction when CRP was added to their model Arts et a0al [] investigated the roles of SCORE FRS RRS and QRISK2 in RA patients Out of these four models SCORE FRS and RRS underestimated CVD risk in RA patients whereas the QRISK2 reported an overestimation The AUC ranged between and for the four risk models A similar study by Arts et a0al [] investigated the performance of the original recalibrated and improved version of SCORE calculators to predict the CVD risk in RA patients The AUC values for these scores were CI and All these three scores underestimated the CVD risk in RA patients In short even after the SCORE was redesigned using the RAbased risk factors it did not result in an adequate CVD risk assessment In another study by Crowson et a0al [] of RA patients a CVDrisk prediction model was developed that reported better performance AUC compared with conventional risk calculators such as FRS AUC ACCAHA AUC SCORE AUC and QRISK2 AUC Furthermore conventional risk calculators either overestimated or underestimated CVD risk in RA patients Wahlin et a0al [] compared the expanded risk score ACCAHA risk score and a modified version of ACCAHA with a multiplier of for a CVD risk assessment of 0c Rheumatology International RA patients The authors also reported an underestimation of CVD risk by all calculators However the discrimination ability was slightly better since AUC for ERSRA risk was compared to AUC of for two variants of ACCAHAThe overall trend of all these risk prediction algorithms developed for general and RA cohorts indicates a poor CVD risk assessment in patients with RA One possible reason for such poor performance is the paradoxical behavior of some of the risk factors such as lipids and body mass index Another potential reason for this outcome is the inclusion of risk factors that do not provide complete information about the CVD risk profile in RA patients [] Corrales et a0al [] indicated a high prevalence of carotid atherosclerosis plaque in patients with lowCVD risk This observation demonstrated the limited ability of conventional risk factorbased algorithms to improve the CVD risk assessment process which may be improved using imaging modalities Therefore there is still room to develop more accurate automated and reliable risk calculators for RA patients by exploring and including nontraditional risk factors such as genetic biomarkers inflammatory biomarkers or imagebased atherosclerotic plaque phenotypes in the risk prediction algorithmCarotid ultrasound atherosclerosis imaging for a0CVD risk assessment in a0RA patientsImaging modalities are becoming essential for the visualization of atherosclerotic plaque and CVD risk assessment in RA patients [] Noninvasive imaging modalities such as computed tomography magnetic resonance imaging ultrasound and positron emission tomography are currently used to assess carotid atherosclerosis in RA patients [] MRI is used to measure the plaque composition including calcification lipidrich necrotic core and fibrous cap thickness [] Computed tomography is generally used to determine carotid artery stenosis [] Ffludeoxyglucosepositron emission tomography FDGPET is a nuclear imaging modality that quantifies the inflammation in carotid atherosclerotic plaque [] Noninvasive carotid ultrasound is a commonly adopted imaging modality that can capture morphological variations in the atherosclerotic plaque quantified using carotid intimamedia thickness cIMT carotid intimamedia thickness variability IMTV and plaque area [] When compared with other noninvasive counterparts carotid ultrasound is less expensive and easier to use [ ] Therefore the scope of this review is restricted to the use of carotid ultrasound for CVD risk assessment in RA patients The automated cIMT and carotid PA are considered surrogate markers of coronary artery disease and widely used for CVDstroke risk assessment []Several studies have shown a high prevalence of increased cIMT and carotid plaque in RA patients [] Studies have also demonstrated the significant association between these carotid atherosclerosis biomarkers and RAspecific markers of inflammation such as ESR CRP and IL6 [] Table a0 provides some of such studies that link both carotid atherosclerosis and RA using two sets of biomarkers ie imagebased phenotypes and inflammatory biomarkers One common observation from these studies is that patients with RA show an elevated cIMT and carotid plaque area compared with nonRA cohorts row R2R4 of Table a0 [ ] This association between carotid atherosclerosis and RA also seems independent of the three carotid artery segments common carotid artery carotid bulb and internal carotid artery from where the cIMT or plaque was measured [ ] However several studies have reported more aggressive atherosclerotic plaque formation in the carotid bulb segment when compared to other arterial segments [] The higher plaque prevalence in the carotid bulb is a consequence of turbulent blood flow and reduced shear stress which leads to endothelial dysfunction [ ] This observation of higher plaque in a bulb has also been confirmed in RA patients [] Figure a0 shows carotid ultrasound scans for RA Fig a03a b and nonRA patients Fig a03c d The lefthand side panel of Fig a03a c shows the raw carotid ultrasound scans measured using a Bmode ultrasound scanner The broad usage of carotid ultrasoundbased phenotypes and their significant association with RAspecific Similarly the righthand side panels of Fig a03b d show the processed scans tracking morphological variations in the carotid atherosclerotic plaque for the quantification of cIMT and plaque area The cIMT and plaque area are both greater in RA patients than in nonRA patientsAnother important observation from Table a0 is the significant association between carotid atherosclerotic biomarkers and RAspecific inflammatory markers such as ESR CRP and IL6 [ ] ESR is a relatively inexpensive measure of inflammation in RA patientstherefore several studies have used ESR for CVD risk assessment [] Some of such studies are listed in Table a0 All these studies indicated a substantially higher CVD event rate in patients with elevated ESR levels Besides ESR studies have also suggested the use of other popular RAspecific inflammatory markers such as CRP or hsCRP and IL6 for the improvement in the CVD risk assessment [ ] Furthermore these RAspecific inflammatory markers are also associated with the annual progression of cIMT [ ] which is a prominent surrogate marker of cardiovascular events In a study with RA patients Kaseem et a0al [] demonstrated the association of ESR CRP and IL6 with carotid atherosclerosis with significant odds ratios p of and respectively 0c hti foTM wdetaicossa era PCIc PRCdna RSE ecneserp dna TM 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RSE sa eht tciderp ot desuhcus eranedrub euqalp ditorac srekramyrotammaflnI ni tnatropm DACgnitciderp rofi osla era TMIc dna thgiehPC ecneserp eht PC fodnoyeBstneitapAR mm a0 ¥ thgieh mm a0 ¥TMIc PC roF hgihPRCsh nietorpLDHdoolb cilotsaid loretselohc PBDnietorpopil erusserp evitcaer ytisnedwol cLDL loretselohc latot CPRC etar noitatnemides etycorhtyre RSE sgurdnoitacrufibFIB citamuehritna CT doolb cilotsys PBS yrotsih ylimaf HF sutillem setebaidMD gniyfidomesaesidnoisnetrepyhNTH yretra ditorac lanretni ditorac mumixam xamTMIc ssenkciht aidemamitni ditorac TMIc esaesid yretra yranoroc DAC esaesid ralucsavoidrac DVCACI yretra xedni ssamydob IMB sDRAMD rotcaf diotamuehr FR nikuelretni LI nietorpditorac nommoc ACC sitirhtra diotamuehr AR euqalp ditorac PC stneitap fo rebmunN rebmun laires NS edirecylgirt GT loretselohc evrucehtrednuaera CUA ssenkciht aidemamitni evitcaer Cytivitisnesnietorpopil ytisnedhgih oitar sddoRO erusserp cRheumatology Internationalinflammatory markers has also enabled their inclusion in the CVD risk prediction calculators [ ] Recently several CVDstroke risk prediction models have been developed that have combined the effect of conventional risk factors and the automated carotid atherosclerosis biomarkers [ ] These risk prediction models reported a better performance in identifying high CVD risk patients compared with current standardofcare risk calculators However such socalled integrated risk prediction models were developed for the general population They were based on the annual progression rates of carotid atherosclerotic biomarkers and conventional risk factors [] Therefore given the progression rates of cIMT and PA due to the RAspecific inflammatory markers such models can be updated and might be useful for CVD risk assessment in RA patientsArtificial intelligence in a0CVDstroke risk assessmentArtificial intelligence AI is expeditiously changing the landscape of the global healthcare system and assisting the healthcare workforce in clinical decisionmaking [] Machine learning ML and deep learning DL are the two common branches of AI that have broad ranges of applications in almost every medical imaging sector eg classification and plaque characterization for stroke risk assessment [] thyroid cancer characterization [] liver cancer diagnosis [] prostate cancer diagnosis ovarian cancer diagnosis [] lung cancer detection [] brain tumor classification [] and heart disease prediction and disease classification [ ] During the recent global pandemic of coronavirus diseases AI is providing promising results in the diagnosis of patients with the help of several imaging techniques such as computed tomography [] and Xrays []Since this review is on CVDstroke risk assessment we have summarized several studies that have used MLbased algorithms for CVDstroke risk assessment Table a0 All of these studies follow a supervised learning approach in which the MLbased classifier is trained to identify the correct output labels using input risk factors or features and predefined gold standards or labels Figure a0 shows the generalized framework of supervised MLbased CVD risk assessment In the case of CVD risk assessment the gold standard can be the primary endpoints such as presence or absence of cardiovascular events or surrogate endpoints such as cIMT PA and CAC score or a combination of these risk factors [ ] Several types of input features can be used to train the AIbased algorithms They can be traditional risk factors imagebased phenotypes grayscale image features or statistically derived features Once the offline ML classifier is trained using these features and gold DAC rof RO roF gnikomS MD NTH PBD PBS IMB egAaimedipilrepyH thgiehPCdna TM Ic ± ][ nosetnavSR 0cRheumatology International Fig Carotid ultrasound image of the common carotid artery for control patientsstandard its coefficients are then used in the online ML system to predict the out risk labels Online ML systems do not require a gold standard to make the final risk classification All the studies provided in Table a0 used this approach for CVD risk assessment Unlike MLbased algorithms DLbased models such as convolutional neural networks do not require input features beforehand Instead such algorithms automatically learn their offline coefficients from the input image datasets [] Currently AIbased techniques are used in the diagnosis of RA [] the identification of RA disease severity [] the classification of several RA synovial tissues [] and mortality prediction due to RA [] Although MLbased algorithms are used in the RA field no efforts have been made to assess the CVD risk in RA patients using such automated intelligencebased paradigms MLbased algorithms have been used to perform CVD risk assessments in nonRA populations and reported a better performance in identifying highrisk CVD patients when compared with the current standard of care conventional risk calculators [ ] Patients with RA experience more atherosclerotic plaque in the carotid artery which might lead to cardiovascular events [] In recent years several studies have demonstrated a better stroke risk assessment using MLbased strategies [] and DLbased strategies [] Besides all these studies attempts can be made to develop more accurate CVD risk prediction tools for RA patients using AI techniques Figure a0 conceptualizes several components required for CVD risk assessment in RA patients The AIbased CVD risk assessment for RA patients can be made possible by combining several types of risk factors such as patients demographics physiological parameters behavioral risk factors imagebased phenotypes and most importantly RAspecific inflammatory markers This combined set of features can be used as inputs along with the gold standard to identify what CVD risk category RA patients belong to As such both ML and DLbased systems can be employed to performed CVD risk assessment in patients Because of the significant association between carotid atherosclerosis and RA researchers can conduct a pilot study with cIMT and plaque areas as the surrogate markers for CVD risk assessmentSummaryIn this review we provided several | 2 |
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes | 2 |
Breast cancer BC is the most common malignant tumour in women worldwide and one of the most common fataltumours in women DeltaNotchlike epidermal growth factor EGFrelated receptor DNER is a transmembraneprotein involved in the development of tumours The role and potential mechanism of DNER inepithelialmesenchymal transition EMT and apoptosis in BC are not fully understood We ï¬nd that DNER isoverexpressed in BC tissue especially triplenegative breast cancer TNBC tissue and related to the survival of BC andTNBC patients In addition DNER regulates cell EMT to enhance the proliferation and metastasis of BC cells via theWntcatenin pathway in vitro and in vivo Moreover the expression levels of catenin and DNER in BD tissue arepositively correlated The simultaneously high expression of DNER and catenin contributes to poor prognosis in BCpatients Finally DNER protects BC cells from epirubicininduced growth inhibition and apoptosis via the Wntcatenin pathway In these results suggest that DNER induces EMT and prevents apoptosis by the Wntcatenin pathway ultimately promoting the malignant progression of BC In our study demonstrates thatDNER functions as an oncogene and potentially valuable therapeutic target for BCIntroductionBreast cancer BC is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 BC treatments can beused to improve patient outcome3 However tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure Therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of BC is urgentCorrespondence Si Sun karensisi126com or Shengrong Sun sun137sinacom1Department of Breast and Thyroid Surgery Renmin Hospital of WuhanUniversity Wuhan Hubei China2Department of Pathophysiology Wuhan University School of Basic MedicalSciences Wuhan Hubei ChinaFull list of author information is available at the end of the These authors contributed equally Zhong Wang Zhiyu LiEdited by S TaitTumour EMT is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4 EMT a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8 Recent studies upon activation of theclassical Wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating EMT10 Therefore maintaining cateninactivity is important for the Wntcatenin pathway andtumour progressionDNER a neuronspeciï¬c transmembrane protein foundin a variety of peripheral cells11 is a member of theatypical Notch ligand family and binds to Notch1 receptor1115 DNER is expressed at abnormally high levels in The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 Nevertheless the precise function and underlying molecularmechanisms of EMT and chemosensitivity in BC areunclearIn this study we have revealed the previously unrecognized role of DNER in cancer progression EMT andthe apoptosis of BC cells Furthermore we investigatedthe expression of DNER and its relationship with survivalin BC and TNBC patients In addition we have providedevidence for the correlation between DNER and cateninand the prognostic value of the highlevel expression ofDNER and catenin in BC patients Finally the crucial roleof catenin in DNERinduced EMT and the inhibitoryeffect of DNER on apoptosis have been revealed Takentogether our results elucidate the potential functions andmechanism of DNER in EMT and apoptosis in BC cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of BCMaterials and methodsEthics statementTwo groups of the same human tissue specimens wereacquired from patients of Renmin Hospital of WuhanUniversity who were diagnosed with BC from to One group of specimens was promptly stored at °C for western blotting and PCR analysis The othergroup of specimens was ï¬xed in formalin and parafï¬nizedfor immunohistochemistry IHC All patients did notreceive chemotherapy radiotherapy or immunotherapyThis research was approved by the Ethics Committee ofRenmin Hospital of Wuhan University and informedconsent was obtained from all patientsCell culture and reagentsHuman BC cell lines MCF7 and MDAMB468 cellswere obtained from American Type Culture Collectionand incubated by their corresponding recommendedmethod All celllines were mycoplasmafree by morphological examination and veriï¬ed for their authenticities by STR proï¬ling Epirubicin was purchased fromPï¬zer Pharmaceutical Co Ltd Wuxi China and dissolved in physiological saline CHIR catenininhibitor and XAV939 catenin agonist were purchased from Selleck Shanghai China and dissolvedin DMSO and The stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining The ï¬nal protein staining score was the percentage score multiplied by the intensity score ï¬nalprotein staining scores were divided into three categoriesas follows negative low expression and high expressionsiRNA and plasmid transfectionscrambleDNER siRNA ²GCUUUGCCAGUCCAAGAUUTTsiRNA ²UUCUCCGAACGUGUandCACGUTT were synthesized from GenePharma CoShanghai China FLAGDNER and FLAGNC werepurchased from GeneChem Co Shanghai China Whencells in a sixwell plate had grown to the appropriatedensity siRNA and plasmids were transiently transfectedwith Lipofectamine3000 Invitrogen USA and RNAiMAX Invitrogen USA respectively according to themanufacturers instructions After h of transfection thecells were used for subsequent experimentsqRTPCRTotal RNA from tissue specimens and cell samples wasextracted by using TRIzol Invitrogen USA according tothe protocol and then reverse transcribed to cDNA usinga TransScript FirstStand cDNA Synthesis Kit TaKaRaJapan qRTPCR was implemented by using SYBR GreenMastermix TaKaRa Japan with an ABI 7900HT RealTime PCR system USA The primer sequences areshown in Supplemental Table Cell Counting Kit CCK8 assayAfter a series of interventions equal numbers of BCcells were plated into 96well plates and cultured for days Ten microlitres of CCK8 CK04 Dojindo Japansolution was added to each well and the cells wereincubated at °C for h The absorbance was determined at nmWound healing assayAfter intervention the cells were seeded into sixwellplates When the cell density exceeded the cells werewashed twice with PBS and scratches were made with ayellow plastic pipette tip Cells were cultured in serumfree medium for h and photographed under amicroscopeImmunohistochemical stainingInvasion assayIHC staining was performed as previously described18The results of IHC staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensityThe percentage of positive cells was scored as follows After a series of treatments à cells in serumfreemedium were plated in the upper chambers of a Transwell apparatus with Matrigel Corning NY USA Medium in the bottom chambers containing FBS servedas an attractant After h of incubation cells that passedOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of through the chamber membrane were ï¬xed with precooled formaldehyde and stained with crystal violetC0121 Beyotime The cells were counted and photographed under a microscopeWestern blottingThe prepared tissue and cell samples were separated byprotein SDSPAGE and transferred to a nitrocelluloseNC membrane The membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °C overnight Afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantiï¬edby ImageJ software W S Rasband Image J NIH Theantibodies used are listed in Supplementary Table Nuclear and cytoplasmic protein extractionNuclear and Cytoplasmic Extraction Reagent P0027was purchased Beyotime Biotechnology The nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsFlow cytometry to detect apoptosisA FITC Annexin V Apoptosis Detection Kit I BDPharmingen USA was used to detect cell apoptosis The cellswere seeded in sixwell plates After a series of interventionscells were processed following the manufacturers protocolFig DNER is upregulated in BC tissues and correlated with poor prognosis in BC and TNBC patients a The expression levels of DNER inluminal A and TNBC tumour tissues compared with adjacent tissue by IHC magniï¬cation à b The mRNA levels of DNER in luminal A and TNBCtumour tissues compared with adjacent tissue c The DNER protein expression in BC tissues and adjacent tissues by western blotting d TheKaplanMeier analysis showed the RFS of BC and TNBC patients with DNER high expression or DNER low expression e The staining of DNER Ecadherin and Ncadherin in BC tissue by IHC magniï¬cation à f Correlation analyses of protein expression levels between Ecadherin Ncadherinand DNER p p vs the control groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of and the cell ï¬uorescence was measured with a FACScan ï¬owcytometer FACScan Becton DickinsonTable Clinicopathological associations of DNERexpression in breast cancerAnimal experimentsTo acquire MDAMB468 cells with DNER stablyknocked down and MCF7 cells stably overexpressingDNER cells were transfected with DNER knockdown andoverexpression lentivirus GeneChem Shanghai Chinaand then selected with puromycin When the transfectionefï¬ciency approached the DNER protein level wasdetected with western blotting All experimental procedures were conducted according to the Regulations ofExperimental Animal Administration issued by the Animal Committee of Wuhan University The mice wererandomly divided into two groups A total of à stable cells in μl PBS were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice BALBc After a certain period ofintervention the mice were sacriï¬ced by anaesthesia andxenografts were removed for weighing and photographing The expression of relative proteins was detected bywestern blotting and IHCFor mammaryfatpad tumour assays we establishedMDAMB231 cells with DNER stably knocked downThe mice were randomly divided into two groups à stable cells were resuspended in a mixture of PBS andMatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice To observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 Ten days after theoperation the mice were sacriï¬ced by anaesthesia and thenumber of metastatic tumours per lung were determinedThe entire lung tissues were ï¬xed with formalin andsectioned for haematoxylin and eosin HE staining todetermine the presence of lung metastasis The entirelung tissues were ï¬xed with formalin and sectionedfor haematoxylin and eosin HE staining to determinethe presence of lung metastasisImmunoï¬uorescenceImmunoï¬uorescence staining was performed as previously described19 In brief after corresponding treatments the cells ï¬xed with paraformaldehyde wereperforated by TritonX for min and blockedwith BSA for h Next the cells were incubated withcatenin dilution overnight at °C and thenincubated for min with 488conjugated antibodyInvitrogen A11034 Finally the slides were stained withDAPI for min The images of sample were analyzed bylaser confocal microscopy Zeiss LSM Statistical analysisStatisticalSPSS software SPSS Inc Chicago IL and GraphPadanalyses were performed usingOfï¬cial journal of the Cell Death Differentiation AssociationVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2Prism GraphPad Software La Jolla CA USA All datawere analyzed with at least three independent experiments and are presented as the mean ± SD A survivalcurve was prepared by KaplanMeier analysis and thelogrank test was used to compare survival differencesbetween groups Pearsons correlation method was used 0cWang Cell Death and Disease Page of Table Clinicopathological associations of DNERexpression in triple negative breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P to analyze the correlation between DNER and cateninA chisquare test was used to analyze associationsbetween DNER expression levels and clinical characteristics Oneway ANOVA was used to compare differencesin three or more groups Differences in which p were considered statistically signiï¬cantResultsDNER is upregulated in BC tissues and correlated withpoor prognosis in BC and TNBC patientsTo determine the role of DNER in development of BCwe ï¬rst measured the expression levels of DNER in BCtissue and matched adjacent normal breast tissue by IHCThe expression level of DNER in BC tissue was markedlyhighertheexpression in TNBC was higher than that in luminal A BCFig 1a We also detected the expression of DNER in BCtissue by PCR the results of which were consistent withthose of IHC experiments Fig 1b To further verifytissue moreoverthan thatin adjacentOfï¬cial journal of the Cell Death Differentiation AssociationDNER expression in BC we utilized western blotting todetect DNER protein expression in BC and adjacent tissues As expected compared with DNER expression inadjacent tissues DNER expression in BC tissues wassigniï¬cantly elevated Fig 1c Furthermore the highestDNER expression level was found in TNBC tissue Theclinicopathological characteristics with different expression of DNER in all BC and TNBC patients were shown inTables and KaplanMeier analysis of RFS showed thatthe group expressing high levels of DNER had a worseprognosis than the group expressing low levels of DNERThe results of survival analysis of TNBC patients were thesame as that of BC patients and TNBC patients had ashorter RFS than BC patients Fig 1d Next to verifywhether the poor prognosis of BC patients caused byDNER is related to EMT we detected the correlationbetween DNER and EMTrelated markers The resultsshowed that DNER expression was negatively correlatedwith the expression of Ecadherin while positively correlated with Ncadherin expression Fig 1e f In addition we found that high expression of mesenchymalmarkers was signiï¬cantly associated with high expressionof DNER in BC through the TCGA database httpgepiacancerpkucn Although the negativecorrelationbetween Ecadherin and DNER in TCGA database wasnot signiï¬cant it also presented a negative trend Supplementary Fig 2A The results therefore suggested thatDNER is highly expressed in BC and that elevated DNERprotein expression contributes to the progression of BCespecially TNBCDNER increases the biological functions of BC cells in vitroTo evaluate the effect of DNER on BC cell proliferationmigration and invasion we used siRNA to suppressDNER expression in both MCF7 and MDAMB468cells Compared with DNER expression in the control andscramble siRNA groups DNER was silenced by almost and in MCF7 and MDAMB468 cells transfected with siRNA respectively Fig 2a b As shown inFig 2c DNER knockdown visibly downregulated thegrowth rate of BC cells by CCK8 assay Next a woundhealing assay was used to evaluate cell migration capacityCompared with wound closure in the scramble siRNAgroup DNER knockdown signiï¬cantly inhibited woundclosure after h in BC cells Fig 2d In addition theTranswell assay revealed that DNER knockdown clearlyreduced BC cell invasion Fig 2e These results suggestthat DNER acts as a cancerpromoting gene in BC cellsTo further conï¬rm the role of DNER in BC progressionDNER was overexpressed by transfection with the FLAGDNER plasmid for h As shown in Supplementary Fig1A DNER was successfully overexpressed in the two BCcell lines In striking contrast with the effects of DNERknockdown the ability of cell proliferation migration and 0cWang Cell Death and Disease Page of Fig DNER knockdown inhibits cell proliferation and metastasis of BC cells a b The knockdown efï¬ciency of DNER in MCF7 and MDAMB cells c Cell growth was measured by CCK8 assay after DNER knockdown in two BC cell lines d Wound healing assay was used to determine themigratory ability of BC cells with DNER knockdown e The invasion capacity of BC cells with knockdown of DNER was conï¬rmed by Transwell assayDown Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after DNER overexpression Supplementary Fig 1BE Taken togetherthese results indicated that DNER plays a crucial role inBC growth and metastatic potentialDNER induces EMT in BC cellsTumour cell EMT promotes the malignant progressionand metastasis of tumour cells10 We next examinedwhether DNER has a regulatory effect on BC cell EMTTo assess this function we detected EMTrelated proteinexpression by western blotting DNER knockdown signiï¬cantly upregulated epitheliallike marker Ecadherinexpression and downregulated mesenchymal marker Ncadherin Vimentin Snail expression Fig 3a b Conversely overexpression of DNER dramatically shown theopposite effect Fig 3c d These results indicate thatDNER drives EMT in BC cells To provide further evidence of this effect of DNER on EMT we suppressedDNER expression and then transfected cells with theFLAGDNER plasmid to restore the DNER protein levelwe then determined whether DNER overexpression couldreverse changes in the expression of EMTrelated proteins As shown in Fig 3e f DNER knockdown alone hadan inhibitory effect on EMT whereas DNER knockdownand FLAGDNER transfection suppressed the effect ofDNER knockdown on Ecadherin and partially restoredthe expression of Ncadherin Vimentin and Snail Theseresults suggest that DNER plays a pivotal role in inducingEMT in BC cellsOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER induces EMT in BC cells a b EMTrelated proteins Ecadherin Ncadherin Vimentin and Snail were detected by western blotting inDNER knockdown cells Right quantitative analysis of the optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actinare shown c d EMTrelated protein levels were measured by western blotting after DNER overexpression in BC cells Right quantitative analysis ofthe optical density ratio of Ecadherin Ncadherin Vimentin and Snail compared with actin are shown e f DNER was overexpressed in DNERknockdown cells and then western blotting detected the expression of EMTrelated proteins The values are the mean ± SD from three independentexperiments p p p vs the corresponding groupDNER activates the Wntcatenin signalling pathway andis positively correlated with cateninPrevious reports have shown that the Wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and EMT2021 Therefore we examined whether DNER mediates the canonical Wntcatenin signalling pathway As shown in Fig 4a b compared withcontrol cells in DNER knockdown cells the protein levelsof Notch1 pGSK3 and catenin were increased andthose of GSK3 were unchanged Conversely DNERoverexpression dramatically shown the opposite effectNext we investigate whether there is a relationshipbetween Notch signal and catenin in the case of DNERoverexpressioncells weIn DNERoverexpressingknocked down Notch1 and found that catenin expression was decreased compared with DNER overexpressionalone Supplementary Fig 2B Notch1 functioned as animportant role in the Wntcatenin pathway and theactivation of Notch1 was positively related to the nucleartranslocation of catenin22 Theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours We assessed the effectof DNER knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of DNER the levels of nuclear catenin and Snailwere reduced in BC cell lines Fig 4c and SupplementaryFig 2C The nuclear location of catenin detected byimmunoï¬uorescence showed the same results as thoseOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER activates the Wntcatenin signalling pathway and is positively correlated with catenin a b Western blotting detected theexpression of Notch1 pGSK3 GSK3 and catenin after DNER knockdown or DNERoverexpressing in BC cells c Total proteins catenin andSnail nuclear proteins catenin and Snail in DNER knockdown cells were assayed with western blotting d The mRNA levels of Survivin cMyc andLEF1 were detected by qRTPCR e The staining of DNER and catenin in BC tissue by IHC magniï¬cation à f Correlation analyses of proteinexpression levels between DNER and catenin g KaplanMeier survival analysis of BC patients was performed with DNERHighcateninHigh andDNERLowcateninLow expression The values are the mean ± SD from three independent experiments p p vs thecorresponding groupdetermined by western blotting Supplementary Fig 2DTo further conï¬rm the decrease in nuclear cateninaccumulation following DNER knockdown we examinedthe expression levels of catenin downstream targetgenes in BC cells by PCR Consistent with the westernblotting results the mRNA expression levels of SurvivincMyc and LEF1 were signiï¬cantly downregulated uponDNER knockdown Fig 4d These data indicated thatDNER knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe Wntcatenin signalling pathwayTo verify the relationship between DNER and cateninwe measured the protein expression levels of DNER andcatenin in BC tissues IHC showed that catenin washighly expressed when DNER was overexpressed whilecatenin levels were low when DNER was knocked downFig 4e Interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of DNER Fig 4f We also found a strongpositive correlation between DNER expression andnuclear catenin expression Supplementary Fig 2EFurthermore immunoï¬uorescence analysis showed thatDNER overexpression promoted more nuclear accumulation of catenin in BC cells Supplementary Fig 2FFinally KaplanMeier analysis showed that the prognosisof BC patients with high levels of DNER and cateninwas worse than the prognosis of BC patients with lowlevels of both DNER and catenin Fig 4g In additionOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Table Clinicopathological associations of both DNERand catenin expression in breast cancerVariablesLowN HighN P valueAge at diagnosis years¤GradeWellModeratelyPoorlyTumour size cm¤Lymph node metastasisNegativePositiveVascular invasionNegativePositiveERNegativePositivePRNegativePositiveHER2NegativePositiveKi67 ¥ RecurrenceNoYes P values calculated by logrank testing bold if statistically signiï¬cant P ER oestrogen receptor PR progesterone receptor HER2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both DNER and catenin and BCpatient clinicopathologic features as shown in Table These data suggest a strong correlation between theexpression of DNER with that of catenin and high levelsof DNERcatenin with poor prognosis in BCOfï¬cial journal of the Cell Death Differentiation AssociationThe Wntcatenin signalling pathway is involved in DNERinduced EMT and prometastatic phenotypesTo determine whether the Wntcatenin pathwayfunctions in DNERinduced EMT we assessed whetherCHIR a speciï¬c Wntcatenin pathway activator23 and XAV939 a Wntcatenin pathway inhibitor24 could reverse the effect of DNER overexpressionand DNER knockdown in BC cells Catenin levels in thetwo BC cell lines were signiï¬cantly elevated after CHIR treatment and markedly suppressed after XAV939treatment Fig 5a b Compared with DNER knockdownalone levels of the EMTrelated proteins were dramatically exhibited the opposite effect after of the treatment ofDNER knockdown cells with CHIR Fig 5a Thetreatment of DNERoverexpressing cells with XAV939clearly show similar results Fig 5b These ï¬ndingsindicated that CHIR partly rescued the inhibitoryeffect of DNER knockdown on EMT progression and thatXAV939 suppressed the activation of EMT induced byDNER overexpression To investigate the role of the Wntcatenin pathway in DNERmediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in DNER knockdownor DNERoverexpressing cells respectively Consistentwith the effects of Wntcatenin pathway activation andinhibition on EMT in the presence of CHIR theproliferation migration and invasion of DNER knockdown cells were clearly elevated Fig 5c e f Similarlyinhibition ofin DNERoverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion Fig 5d g h Altogether these data suggestedthat catenin is indispensable for DNERinduced BC cellEMT and prometastatic phenotypescatenin by XAV939DNER enhances the tumorigenic and metastatic ability ofBC cells in vivoTo verify our results in vitro we next examined the roleof DNER in vivo To that end MDAMB468 cells inwhich DNER was stably knocked down and MCF7 cellsstably overexpressing DNER were successfully establishedto use to establish xenograft models in mice Fig 6a b fg After a period of time the xenografts were removedphotographed and weighed DNER knockdown signiï¬cantly inhibited tumour size and weight comparedwith those in NC group Fig 6c d Consistent with theeffect of DNER knockdown xenografts from DNERoverexpressing group were larger and heavier than thosefrom NC group More importantly XAV939 reversedchanges in the size and weight of xenografts Fig 6h iThe DNER catenin cMyc and Snail protein levels inxenograft tissue were measured to conï¬rm the upregulation and downregulation by western blotting Fig 6e jSupplementary Fig 3A Moreover IHC results found 0cWang Cell Death and Disease Page of Fig The Wntcatenin signalling pathway is involved in DNERinduced EMT and metastasis a b The expression of EMTrelated proteinsand catenin were detected by western blotting in DNER knockdown or DNERoverexpressing cells with CHIR μM h or XAV939 μM h treatment respectively c d Cell growth was measured by CCK8 in BC cells treated as described above e g Wound healing assay was used toexamined migration ability in BC cells treated as described above f h Transwell assay showed the cell invasion abilities in BC cells treated asdescribed above Right Quantitative analysis of invasion ratio was shown The values are the mean ± SD from three independent experiments p p vs the corresponding groupthat DNER knockdown reduced nuclear location ofcatenin while DNER overexpression promoted thisnuclear translocation effect Supplementary Fig 3C Inaddition as shown in Supplementary Fig 3A C thewestern blotting and IHC results showed that DNERimpacted the tumour growth in vivo was related to thelevel of Ki67 which is consistent with the positive correlation between DNER expression and ki67 expression inBC patients of TCGA database Supplementary Fig 3BTo explore the role of DNER in BC metastasis to lungMDAMB231 cells with stably DNER knockdown wassuccessfully established Fig 6k As shown in Fig 6l theOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig DNER enhances the tumorigenic ability of BC cells in vivo a f k The transfection efï¬ciency of DNER knockdown or expression in MDAMB468 MCF7 or MDAMB231 cells respectively b g The knockdown or overexpression efï¬ciency of DNER in MDAMB468 cells or MCF7 cellsrespectively c h The xenograft pictures of shDNER and NCDNER in MDAMB468 cells n d i Comparison of tumour weights from variousgroups e j The expression of DNER and catenin in xenograft tissue by western blotting h The xenograft pictures of NCDNER group OEDNERgroup and OEDNER treated with XAV939 group in MCF7 cells n l Schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of BC m Bright imaging of the lungs metastasis left and quantiï¬cation of the metastases tumour right generated by MDAMB231cells n p vs the corresponding groupOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of Fig See legend on next pageOfï¬cial journal of the Cell Death Differentiation Association 0cWang Cell Death and Disease Page of see ï¬gure on previous pageFig DNER reduces the chemosensitivity of BC cells to epirubicin in vitro a Cell proliferation was detected by CCK8 after treated withdifferent concentrations of epirubicin in two BC cell lines b c DNER was analyzed by western blotting in BC cells treated as described above Rightquantitative analysis of the optical density ratio of DNER compared with actin are shown d Expression of epirubicininduced DNER was detectedby PCR e Cell viability was assessed by CCK8 after DNER knockdown treated with epirubicin or not f Analysis of apoptosis with FACS in MDAMB cells treated as described in e Right Quantitative analysis of apoptosis ratio g The expression of PARP was detected by western blotting in BCcells treated as described above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown h Cell growthwas measured by CCK8 after DNER overexpression treated with epirubicin or not i Analysis of apoptosis with FACS in MDAMB468 cells treated asdescribed in h Right Quantitative analysis of apoptosis ratio j The expression of PARP was detected by western blotting in BC cells treated asdescribed above Right quantitative analysis of the optical density ratio of cPARP compared with actin are shown The values are the mean ± SDfrom three independent experiments p p p vs the corresponding groupcorresponding treated MDAMB231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 Lung metastasis was observed in each group after days Brightï¬eldpicture demonstrated that more lung metastasis wasfound in the NCDNER group compared with the shDNER group Fig 6m Similar t | 2 |
gastroenteropancreatic neuroendocrine neoplasms gep nens as well as neuroendocrine tumors gep nets are heterogeneous tumors that originate from peptidergic neurons and neuroendocrine cells previously described as carcinoid tumors in most nets are indolent tumors compared with other epithelial malignancies however they are reported to have the potential to metastasize even in well differentiated tumors and are resistant to therapies1 data from the surveillance epidemiology and end results seer database indicate that the incidence of nets has increased significantly approximately times reaching casesyear of which gep nets account for approximately to of all nets and the correspondence qiang feng department of colorectal surgery national cancer centernational clinical research center for cancercancer hospital chinese academy of medical sciences and peking union medical college no panjiayuan south road chaoyang district beijing peoples republic of china email fengqiang2008vipsinacomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars256723 cancer management and research wu this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cwu dovepressincidence and prevalence of colorectal nets are inferior only to those of colorectal adenocarcinoma146 in addition the tumor sites varied markedly by race with the incidence of rectal nets among asian populations increasing from per in to per in which was among white populations147 similarly the incidence of rectal neuroendocrine tumors rnets grew fastest among all nets by approximately times compared with the incidence in accounting for of all nets which makes it the second most common net in china8significantly higher than that for localized colorectal nets endoscopic resection including endoscopic mucosal resection emr and endoscopic submucosal dissection esd and surgery including transanal excisions as well as surgical resections are both effective methods for metastatic tumors somatostatin analogs ssas radiation radiofrequency ablation rfa chemotherapy targeted therapy and peptide receptor radionuclide therapy prrt are all alternatives however the 5year survival rate of nets with lymph node metastasis lnm or distant metastasis is still disappointing with fiveyear overall survival rates of approximately and respectively6911the prognostic factors of colorectal nets have been explored by numerous studies tumor stage location size grade lymphovascular invasion and the status of resection margins are major factors that have been reported to be associated with lnm and poor prognosis12 however these studies were highly heterogeneous which affected the accuracy of the metaanalysis and the effectiveness of these scurrently controversies remain in the treatment of colorectal nets experts from the chinese society of clinical oncology csco agreed that colonic nets greater than cm and less than cm could be completely resected endoscopically when the t stage was less than t2 but the national comprehensive cancer network nccn recommends that these tumors be treated by surgery in accordance with the guidelines for colon adenocarcinoma13the aim of this study was to evaluate the outcomes of colorectal nets explore the risk factors for lymph node metastasis in colorectal nets and identify the prognostic factors for survival outcomesmethodsclinical data collectionbetween and a total of consecutive patients received treatments for colorectal nets in our center we constructed a database of retrospectively collected data from patients medical records including clinical characteristics pathological reports recurrence and survival during the followup periodfor radical resection with lymph node dissection lymph node metastasis was detected by pathological evaluation for local excision such as endoscopic mucosal resection emr endoscopic submucosal dissection esd or transanal excision tae lnm was evaluated through computed tomography ct or magnetic resonance imaging mri before the treatment and during the followup periods the diagnosis of a metastatic lymph node was based on the following criteria size criteria short axis diameter of lymph nodes was greater than mm for round lymph nodes and greater than mm for ovoid lymph nodes morphological abnormalities irregular contour and margin unclear border heterogeneous internal echoes or signal intensity1617 the tumor diameter refers to the longest diameter of the tumor according to pathology reports for patients with distant metastases tumor diameter was determined by endoscopic findings before treatmentpathological diagnosisthe tumor stage was classified according to the american joint committee on cancer ajcc cancer staging manual 7th edition and 8th edition1819 and the tumor grade was classified according to the classification20 for patients before we revised their pathology results and found that they were all neuroendocrine carcinomas necs therefore we classified them as having g3 grade tumors the mitosis count n23 was expressed as the number of mitotic cells in ten highpower fields hpfs from hematoxylin and eosin hestained slides examined with microscopy according to enetswho guidelines g1 grade mitotic image hpfs g2 grade mitotic image hpfs and g3 grade mitotic images2010 hpfs the ki index was calculated as the percentage of cells labeled by immunohistochemistry according to enetswho guidelines g1 grade ki67 positive index g2 grade ki67 positive index to and g3 grade ki67 positive index20 the expression levels of chromogranin a cga n101 and synaptophysin syn n109 were scored according to the percentage of positive cells and the intensity of cell staining the positive cell percentage score was based on the following system points no positive cells point positive cells accounting for to points positive cells accounting for to points submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu positive cells accounting for to and points positive cells accounting for to the positive cell staining intensity score was based on the following system points negative point weakly positive points moderate positive and points strong positive the two scores were multiplied together points for negative to points for weak positive to points for moderate positive and to points for strong positive inclusion criteriapatients who were treated in our center for localized and metastatic colorectal nets from to exclusion criteria patients who had colorectal nets combined with other malignancies patients for whom the pathological diagnosis was mixed adenoneuroendocrine carcinoma patients for whom there was insufficient clinical inappropriate pathology reports information or from outside hospitalsrisk factors for lymph node metastasis and prognostic factors related to survival were investigated in all patientsthe primary endpoint was progressionfree survival pfs which was defined as the interval between initial treatment and the first documentation of disease progression or deathstatistical analysisstatistical analysis was performed using spss for mac version spss chicago il usa continuous data are described as means±sds in this study the risk factors for lnm were assessed using pearsons Ï2 test in univariable analysis and logistic regression analyses in multivariable analysis the 5year overall survival os and progression free survival pfs were analyzed with the kaplanmeier method variables were compared with the log rank test and the multivariable analysis for survival outcomes was conducted using the cox proportional hazards model statistical significance was accepted for p values female ratio was the frequencies of grade g1 g2 and g3 nets were and respectively of the patients patients were resected locally by emr by esd and by transanal excision in addition nets were surgically resected including radical resections multivisceral resections and palliative resections due to distant metastasis the remaining patients were treated by systemic treatment due to distant metastasis the most commonly used chemotherapeutic regimen in our center was the ep regimen etoposide and cisplatin as the firstline chemotherapy and the secondline chemotherapy was variable and included the xelox regimen oxaliplatin and capecitabine the folfox regimen oxaliplatin calcium folate and 5fu and everolimus temozolomide and tegafurgimeraciloteracil and combinations between them the tumor diameter was less than cm in patients and the distance from the anal verge was less than cm in patients lnm was found in cases and distant metastasis occurred in patients two patients had radiologically determined lnm after tae in the followup period and one of them went on to undergo radical surgery the other patient was also found to have liver metastasis therefore he was treated with chemotherapy the clinical and histopathological characteristics are summarized in table risk factors for lnmthe risk factors for lnm through univariate analysis were tumor location in the colon p0001 tumor diameter ¥ cm p0001 t stage p0001 tumor grade p0001 and the positive degree of syn p0012 and cga p0049 table in multivariable analyses tumor diameter ¥ cm or ci p0040 and tumor grade g3 or ci p0001 were independent risk factors for lnm in colorectal ntes tumor location in the colon or ci p0083 and tumor grade g2 or ci p0066 might be independent risk factors for lnm even though the p value was greater than table resultsclinical and histopathological characteristics figure a total of patients were included in our study figure the age of the patients was ± years and the male risk factors for survival outcomesthe median followup period was months range months a total of patients died in this cohort in patients with distant metastasis before treatment patients died during chemotherapy cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepressfigure flowchart of the selection of patientspatients died after multivisceral resections and patients died after palliative resections due to tumor progression in patients without distant metastasis before treatment patients died due to the recurrence of distant metastasis at the liver peritoneum lung pleura and brain and patient died of severe lung infections the 5year progressionfree survival pfs and overall survival os rates of all patients were and respectively the prognostic factors for the 5year pfs and os rate in all patients were age neoadjuvant chemotherapy tumor diameter tumor location tumor grade lnm cga level and treatment method table in the multivariable analysis age ¥ hr ci p00020001 and lnm yes hr ci p00180025 were independent risk factors for 5year pfs and os the cga level [moderate positive hr ci p0010 and strong positive hr ci p0007] were independent risk factors for 5year pfs tumor diameter ¥ cm hr ci p0063 and tumor grade g3 hr ci p0090 were independent risk factors for 5year pfs even though the p value was greater than table comparison of two age groupsin univariable analyses patients were in the 65year group and patients were in the ¥65year group the proportion of tumors with a diameter ¥ cm was significantly higher in the ¥65year group than in the 65year group vs p0016 there were also significantly more patients with lnm in the ¥65year group vs p0041 for t stage the proportion of earlystage t1 patients in the ¥65year group was significantly less than that in the 65year group although p was vs p0086 for treatments there were significantly more patients who were treated with systematic chemotherapy in the ¥65year group vs p0040different operative methods for t1n0m0 colorectal netsthere was no significant difference in tumor grade tumor location surgical margin relapse or 5year os except for tumor diameter p0012 the diameters of tumors resected by emr esd and transanal excision were ± cm ± cm and ± cm respectively table submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table clinical and histopathological characteristics of colorectal nets n135table continued variablesage¥sexmalefemalebmi¥smoking historydrinking historyfamily cancer historyyesnoneoadjuvant chemotherapyyesnoneoadjuvant radiotherapyyesnodistance from the anal verge cm¥tumor locationrectumcolonappendixtreatmentemresdtransanal excisionradical resectionmultivisceral resectionpalliative resectionsystemic treatmenttumor diameter cm¥t stage after chemotherapyxn variableslnmnegativepositivedistant metastasisnegativepositivetumor gradeg1g2g3cga n101negativesyn n109negativen kaplanmeier survival curveskaplanmeier survival curves for os and pfs according to tumor grade diameter location and cga level are shown in figures discussionnets have a relatively good prognosis with a median os time reported to be years months and a 5year os rate of however the survival outcomes varied significantly at different stages of nets the 5year os rate of stage i and ii tumors was reported to be as high as and but dropped to and for stage iii and iv tumors respectively22 according to epidemiological data from the seer database and gkr joint cancer registry the 5year os rates of lymph node metastases stage iiib and distant metastases stage iv are and respectively1610 the 5year os rates of all patients and patients without distant metastasis were and respectively and the 5year pfs rates were and respectively lymph node metastasis is the most important factor that determines the prognosis of nets and the prediction of lymph node continuedcancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable univariable analysis for risk factors for lymph node metastasis n table multivariable analysis for risk factors for lymph node metastasis n135lnm n Ï2 valuep valuen hr cip valueage¥tumor diameter¥ tumor locationrectumcolonappendix tumor gradeg1g2g3t staget0t1t2t3t4cganegativesynnegative notes by logistic regression analyses p values abbreviation hr hazard ratiomost nets are at the g1 phase and g2 or g3 phases account for only to of all nets and have been reported to be risk factors for lnm by numerous studies1223 a multicenter clinical study in china showed that pathological type g3 nec is an independent risk factor affecting the prognosis of patients with rectal nets p similarly sohn et al3 found that the lnm rate of g1 phase rectal nets was only but it increased remarkably to at the g2 phase in our study our results showed that histological tumor grades g2 p and g3 p0001 were independent risk factors for lymph node metastasis lymph node metastasis occurred in of patients with g3 tumors and with g2 tumors but only with g1 tumors the 5year variablessexmalefemaleage¥bmi¥smoking historyyesnodrinking historyyesnotumor locationrectumcolonappendixtumor diameter¥t staget0t1t2t3t4 tumor gradeg1g2g3cga n101negativesyn n109negative notes by pearsons Ï test p values metastasis is necessary for clinicians to choose a suitable treatment the aim of our research was to explore the predictive factors for lymph node metastasis of colorectal nets and assess the current therapeutic algorithmsubmit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table prognostic factors for survival outcomestable continued variables5year pfs 5year os Ï2 valuep valueg3lnmyesnocga negativesyn negativetreatment variablessexmalefemalebmi¥age¥smoking historyyesnodrinking historyyesnoneoadjuvant radiotherapyyesnoneoadjuvant chemotherapyyesnotumor locationrectumcolontumor diameter cm¥t stagextumor gradeg1g25year pfs 5year os Ï2 value p value continuedemresdtransanal excisionradical or palliativesurgerysystemic treatmentnotes kaplanmeier method and log rank test p values os rate decreased sharply from to when the tumor grade increased from g3 to g1 however tumor grade was not significant in survival outcomes possibly because tumor grade affects survival outcomes indirectly by directly affecting lymph node statustumor size has been reported to be a strong predictive factor for lymph node metastasis previous studies have shown that a tumor less than mm is usually limited to the submucosa with a low metastasis risk of less than and the 5year survival rate can reach approximately to according to the enets guidelines surgical treatment is recommended if rnets are greater than cm g1g3 or are g3 phase with or without metastasis endoscopic resection is feasible when the tumors are less than cm g12 phase and t1 stage15 the treatment of rnets in western countries and in china is similar but there have been controversies regarding the treatment of 2cmsized cnets chinese experts agree that endoscopic resection can be considered for cnets less than cm however there is cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable multivariable analysis for survival outcomes5year pfsos hr cip valueage ¥tumor diameter¥tumor locationrectumcolonappendixlnmnoyescganegativesynnegativetumor gradeg1g2g3neoadjuvant chemotherapynoyestreatmentemresdtaesurgerysystemic treatmentnotes by the cox proportional hazards model p values abbreviation hr hazard ratio0019987800003633e1700003066e29200001826e4500343994900007412e29010015970800001686e30no explicit mention of treatment in the enets guidelines and experts from the nccn recommended surgical resection instead of endoscopic resection1315 in our study survival curves were significantly better p0001 among patients with tumors less than cm figure a tumor diameter greater than cm was an independent risk factor for lnm p0040 table and we believe this was due to the small sample size however patients with tumors less than cm had lnm and patients with tumors less than cm also developed lnm the lnm in small nets might be due to the tumor cells extending to the submucosal layer which has abundant lymphatic vessels for them to spread through previous studies have reported that small nets also have malignant potential26 therefore even if tumor size was submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table comparison of different treatment methods for t1n0m0 colorectal netsemresdtransanal excisiong gradeg1g2g3tumor diameter±±±tumor locationrectumcolonappendixsurgical marginpositivenegativerelapse cases5year os rate notes by anova p values Ï2f valuep valuea strong predictive factor for lnm lnm should not be predicted only by tumor size and further examinations such as eus or ct can help us to evaluate the status of lymph nodes more specificallychromogranin a cga and synaptophysin syn are two neuroendocrine differentiation ned immunohistochemistry markers frequently used in nets in univariable analysis there was a significant difference for colorectal nets with different expression levels of cga in terms of both risk factors for lnm and survival outcomes both p005 in multivariable analysis moderate positive p0010 and strong positive cga p0007 were independent risk factors for 5year pfs which has been proven in a wide variety of retrospective analyses27 in addition prospective clinical trials radiant1 and have been performed to assess the prognostic value of cga in advanced nets and the results showed shorter os for patients with elevated cga31 however the increase in the expression level of cga was not proportional to the increase in the lnm rate or 5year os rate and cga was negative in patients with lnm which may affect the accuracy of the prognostic value of cga lindholm et al35 also found that a relevant portion of nets do not show elevated cga levels the major problem is that several confounding factors can including gastrointestinal and affect cga levels figure a pfs curves according to tumor grade b os curves according to tumor gradecancer management and research submit your manuscript wwwdovepresscom dovepress ab050100150200020406080100progressionfree survivalg1g2g3 0cwu dovepressfigure a pfs curves according to tumor diameter b os curves according to tumor diameterfigure a pfs curves according to tumor location b os curves according to tumor locationfigure a pfs curves according to age b os curves according to agesubmit your manuscript wwwdovepresscom dovepress cancer management and research ababab 0cdovepress wu figure a pfs curves according to cga level b os curves according to cga levelcardiovascular disorders or proton pump inhibitor ppi consumption and a variety of other nontumor reasons36 regarding syn there was only a significant difference in the univariable analysis for lnm previous studies have shown that patients with a low level of synaptophysin had a better os rate than those with a high level however the small sample size limited the accuracy of the results37 based on the findings of this research and previous studies38 it can be suggested that ned might affect the survival outcomes of colorectal net patients and markers especially cga might be suitable for clinicians to predict the prognosis of patientsthe location of the tumor is also an important factor affecting the prognosis and treatment of nets tumors in the colon are more common in necs and generally have a worse prognosis with higher metastatic potential than tumors in the rectum the outcomes of neuroendocrine tumors from the right hemicolon of the midgut and from the left hemicolon from the hindgut are not the same the welldifferentiated biological behavior of the left hemicolon is closer to that of the rectum a recent chinese multicenter study found that more than of colonic nets of midgut origin are necs or mixed adenoneuroendocrine carcinomas manecs39 according to statistics from the seer database the 5year survival rate of patients with rnets is which is significantly higher than that of colonic nets in our study the lymph node metastasis rate in the colon was which was significantly higher than that in the rectum the 5year os rate and pfs rate of individuals with lnm in the appendix and in the rectum were significantly better than those of individuals with lnm in the colon p0005 and p0003 respectively which was consistent with previous studies1 based on the findings of this research and previous studies it can be suggested that colonic nets should be completely resectedin our research the survival outcomes of patients years and older in our study were worse than those of patients younger than years p0001 we also found that tumors from elderly patients ¥ years were larger and more advanced than those from younger patients years both p0001 the reason for the poor prognosis in elderly patients may be that elderly patients have lower tolerance to surgical trauma and side effects of chemotherapy because of their weakened an physiological functions which leads to multiple complications4142 therefore when we encounter elderly patients minimally invasive therapies such as laparoscopic surgery could help reduce surgical trauma and chinese herbs can relieve and reduce the adverse events of chemotherapy43for nets in the colon the recommended treatment varies among different guidelines but surgical resection is generally recommended because of the greater likelihood of malignant behavior than with rectal nets endoscopic resection may be considered if the tumor diameter is less than cm and does not reach the muscularis propria for nets in the rectum eus is required before surgery surgical resection is recommended when the tumor diameter is more than cm g3 grade t3 to t4 stage or when there is peripheral lymph node metastasis when the tumor diameter is less than cm g1 or grade and t1 stage endoscopic resection is feasible in other cases the treatment method is determined according to the depth of tumor invasion assessed by eus21cancer management and research submit your manuscript wwwdovepresscom dovepress 050100150200020406080100months progressionfree survivalnegativeweak positivemoderate positivestrong positivep0023050100150200020406080100months overall survivalnegativeweak positivemoderate positivestrong positivep0038ab 0cwu dovepressthis study has some limitations including its retrospective design and the relatively small number of patients included although lnm should be evaluated after radical resection with lymph node dissection we analyzed the risk factors for lnm by ct or mri in those who underwent local excision before the treatment and during the follow up periods and we believe the results are reliable because this study lasted more than years we could investigate the longterm survival outcomes and prognostic factors after different treatments even with the small number of patients finally further studies should be performed to validate our main sthe clinical and pathological characteristics of rectal and colon neuroendocrine tumors are different | 0 |
" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief models components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbachs alphafor face validity a survey was done on employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tools questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbachs alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartletts research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedont know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring rangecorrect response dont knowresponse incorrect response strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individuals perception of his orher competence to successfully perform a behavior behavioral intention refers to a persons perceived probability orsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items Ï2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartletts test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of educationfemalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that onesintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the researcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med gimenogarca az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarca r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran anz j surg yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis becker mh the health belief model and sick role behavior health educmonogr janz n champion v strecher vj the health belief model k glanz bk rimerjanz nk becker mh the health belief model a decade later health educ qlp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c" | 0 |
" chemotherapy is one of the most commonly used treatments for patients with advanced colon cancer yet the toxicity of chemotherapy agents such as fluorouracil 5fu limits the effectiveness of chemotherapy ginsenoside rg3 rg3 is an active ingredient isolated from ginseng rg3 has been shown to display anticancer effects on a variety of malignancies yet whether rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer remains unknown the present study was designed to ascertain whether rg3 is able to enhance the anticolon cancer effect of 5fu the results revealed that combined treatment of rg3 and fu significantly enhanced the inhibition of the proliferation colony formation invasion and migration of human colon cancer cells sw620 and lovo in vitro we also found that combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway and arrested the cell cycle of the colon cancer cells in g0g1 by promoting the expression of cyclin d1 cdk2 and cdk4 in addition the pi3kakt signaling pathway in colon cancer cells was suppressed by rg3 and 5fu in vivo rg3 synergized the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude mice similarly combined treatment of rg3 and 5fu altered the expression of colon cancer protein in vivo and in vitro collectively the present study demonstrated that ginsenoside rg3 enhances the anticancer effect of 5fu in colon cancer cells via the pi3kakt pathwaycorrespondence to dr xiangbo chen endoscopy center the quanzhou first hospital affiliated to fujian medical university east street licheng quanzhou fujian pr chinaemail coloboyeahnetkey words ginsenoside rg3 colon cancer 5fluorouracil pi3kaktintroductioncolon cancer is a common malignant tumor of the digestive tract located in the colon which mainly occurs at the junction of the rectum and the sigmoid colon statistics show that the highest incidence of colon cancer is in the age group of years the ratio of male to female is and the incidence of colon cancer ranks third among all cases of gastrointestinal tumors the 5year survival rate of patients with colon cancer is approximately yet the 5year survival rate of patients with advanced stage disease is as low as since the early symptoms of patients with colon cancer are not obvious only about of patients can be diagnosed at the early stage of the disease chemotherapy is one of the most important treatments for patients with advanced colon cancer of which fluorouracil fu is the most widely used 5fu inhibits the proliferation invasion and migration of tumor cells by interfering with the nucleic acid metabolism of tumor cells but it is also toxic to normal cells causing serious adverse reactions even endangering the life safety of patients severely limiting its clinical application previous research has shown that 5fu combined with other agents may reduce the required dosage of 5fu consequently reducing the adverse reactions caused by 5fu without affecting or even improving the efficacy of chemotherapy compared with chemical drugs and biopharmaceuticals multicomponent multitarget and less adverse reactions are unique advantages of traditional chinese medicine for the treatment of diseases in patients with colon cancer chinese medicine can improve patient immunity reduce the side effects of radiotherapy and chemotherapy or enhance drug sensitivity inhibiting the expression of oncogenes helps to inhibit the migration of cancer cells and has a good effect on the treatment of colon cancer ginsenoside rg3 rg3 an active ingredient isolated from ginseng is a tetracyclic triterpenoid saponin that inhibits neovascularization induces tumor cell apoptosis and selectively inhibits tumor cell metastasis and enhances immune function previous studies have shown that rg3 exhibits an inhibitory effect on proliferation invasion and migration of human tumor cells such as lung cancer gastric carcinoma and prostate cancer in colon cancer rg3 was found to activate the ampk signaling pathway to accelerate apoptosis in colon 0chong effects of ginsenoside rg3 on colon cancercancer cell line ht29 in vitro and also to block colon cancer progression by targeting inhibition of cancer stem cells and tumor angiogenesis in vivo although numerous studies have shown that rg3 increases the efficacy and decreases the toxicity of chemotherapeutic drugs and suppresses the chemotherapeutic resistance in cancer its combination with chemotherapeutic agent fu to achieve extra benefits in anticolon cancer treatment warrants detailed investigationin the present study the effects of a combined treatment of rg3 and 5fu on the biological properties of sw620 and lovo cells were investigated in vivo and in vitro we found that a combined treatment of rg3 and 5fu not only enhanced the inhibition of colon cancer cell proliferation migration and invasion but also promoted apoptosis of colon cancer cells and arrested the cells in the g0g1 phase in addition it was also found that rg3 could synergize the capacity of 5fu to inhibit the growth of human colon cancer xenografts in nude mouse and the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt pathway in colon cancer cellsmaterials and methodscell lines and agents sw620 ccl atcc american type culture collection manassas va usa and lovo ccl229 atcc cell lines were cultured with dmem medium cat no thermo fisher scientific inc supplemented with fetal bovine serum fbs cat no thermo fisher scientific inc and penicillinstreptomycin cat no thermo fisher scientific inc the cell lines used in the present study were cultured at Ëc with co2rg3 cat no sigmaaldrich merck kgaa and 5fu cat no sigmaaldrich merck kgaa were dissolved in dmso for the cell experiments the diluted culture solution of rg3 or 5fu was dissolved in dmso to achieve the experimental concentration and was administered to the cells for h for animal experiments pbs diluted rg3 or 5fu was dissolved in dmso to the experimental concentration the experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinamtt assay a total of 2x103 cellswell were inoculated in a 96well culture plate containing the indicated medium dmem plus fbs we evaluated the viability of the sw620 and lovo cells by mtt assay in short after h of culture mtt µl mgml which was dissolved in dmso was added to the cells and incubated the cell supernatant was removed and then µl dmso was added after min the optical density od570 was determined using a plate reader elx808 biotek instrumentscell colony formation assay a total of 2x103 cellsml were seeded in 6well plates with ml mediumwell and medium was exchanged once every days cells were routinely cultured for about weeks when visible clones appeared in the well the culturing was stopped the supernatant culture medium was drawn washed with pbs times and fixed with formaldehyde for min the supernatant was drawn stained with crystal violet for min and slowly rinsed with sterile water plates were placed in a sterile purification table and images were captured after drying the relative proliferation was measured by measuring the absorbance at nm using a plate reader elx808 biotek instrumentswestern blot analysis ripa lysate buffer cat no p0013c beyotime institute of biotechnology shanghai china was used to extract total cellular protein and the bca kit cat no p0009 beyotime institute of biotechnology was used to determine the protein concentration then cell lysates of sw20 and lovo cells were separated by sdspage with µg total protein and transferred to a pvdf membrane the following primary antibodies were selected as follows antincadherin antibody ab18203 dilution antiecadherin antibody ab1416 dilution antimmp ab38898 dilution antiactivecaspase antibody ab2324 dilution antiactivecaspase3 antibody ab2302 dilution antiapaf1 antibody ab2324 dilution antipi3kp85 antibody ab191606 dilution antipi3k110 antibody ab32569 dilution antipanakt phospho t308 antibody ab38449 dilution antipanakt antibody ab8805 dilution antipdk1 antibody ab52893 dilution and antigapdh ab9484 dilution the secondary antibody was selected as follows goat antirabbit ab150077 dilution or goat antirat ab150117 dilution the blocking protocol was with skim milk for h at room temperature the primary antibody was incubated overnight at Ëc and the secondary antibody was incubated for h at room temperature the beyoecl plus kit cat no p0018s beyotime was used for the chromogenic protein bands with beckman coulter immunoassay system unicel dxi beckman coulter and imagej v2147 national institutes of health was used for the densitometric analysis of protein bands all antibodies were purchased from abcam unless otherwise statedtranswell invasion experiment the cell density was adjusted to 05x106 cellsml and then the cells were added to a 24well transwell upper chamber corning corning ny usa medium containing fbs gibco thermo fisher scientific inc was added into the lower transwell chamber and the transwell was incubated at Ëc for h the transwell was taken out and the medium was removed it was washed twice with pbs methanol was added and dried after being fixed for min after the membrane was dried it was stained with crystal violet for min and the relative migration was determined by measuring the absorbance at nm using a plate reader elx808 biotek instruments inccell scratch test a total of 5x105 cells were placed in a 6well plate mlwell a scratch was made as far as possible perpendicular to the back of a horizontal line by using tips against a ruler tips should be vertical and cannot be tilted the cells were washed with pbs for three times and the scratched cells were removed and serumfree dmem was added cells were cultured at Ëc in a co2 incubator for h and images were captured in and h using an ckx41 olympus inverted microscope magnification x100 olympus corp 0concology reports figure effect of the combined treatment of rg3 and 5fu on proliferation of colon cancer cells in vitro sw620 and lovo cells were treated with different doses of a rg3 mmoll or b 5fu µmoll and then the mtt assay was used to detect cell viability c and d after treatment with rg3 mmoll or 5fu µmoll or their combination the colony formation of the colon cancer cells was photographed wt group was used as a baseline for cell viability and cell colony formation three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the wt group fu fluorouracil rg3 ginsenoside rg3 flow cytometric analysis cells that had been treated in different manners were collected and precooled ethanol prechilled pbs and waterfree configuration was added at Ëc overnight then the cells were washed with pbs and stained with propidium iodine pi for cell cycle macsquant® analyzer flow cytometer miltenyi biotec was used to detect the cell cycle and the annexin v fitcpi kit invitrogen thermo fisher scientific inc was used for flow cytometry to detect apoptosisanimal experiment human colon cancer cells 5x10602 ml in the logarithmic phase were selected a total of female nude mice weeks of age g shanghai lingchang biological technology co ltd that were adaptive for feeding [room temperature of Ëc half day light and night dark cycle air humidity of ] for one week were selected mice were anesthetized [ sodium pentobarbital mgkg intraperitoneal ip] and then the lateral skin of the nude mice was selected as a cell inoculation site to inoculate 5x10602 ml human colon cancer cells at the logarithmic phase of growth when the tumor tissue grew to a volume of approximately mm3 then the mouse were randomly assigned to the solvent group equal amount of pbs dmso rg3 group mgkg gavage administration once every two days 5fu group mgkg ip injection once every two days and rg35fu group combined rg3 and 5fu group administration after weeks of treatment the mice were sacrificed using cervical dislocation and breathing and heartbeat for min were observed to determine death and tumor tissues were extracted and weighed with an analytical balance bsa124s beijing sartorius instruments ltd beijing china all animal experiments were approved by the ethics committee of quanzhou first hospital affiliated to fujian medical universitystatistical analysis all data are expressed as mean ± standard deviation and spss ibm corp was used to analyze the data student's ttest was used to compare differences between two groups and multiple groups were compared with oneway anova followed by duncan test as a post hoc test p005 was assigned to indicate that a difference was statistically significantresultscombined treatment of rg3 and fu enhances inhibition of cell proliferation after treatment with different doses of rg3 or 5fu mtt assay was used to measure the cell viability the results revealed that the cell viability of sw620 and lovo cells was significantly and gradually decreased with an increasing dose of rg3 thus we chose mmoll rg3 for subsequent experiments fig 1a as shown in fig 1b the proliferative activity of the colon cancer cells in the combined treatment group of rg3 and fu was significantly lower than 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on migration and invasion of colon cancer cells in vitro treatment of colon cancer cells with combined treatment of rg3 mmoll and 5fu µmoll inhibited the migration a and invasion b abilities of the sw620 and lovo cells scale bar µm c western blot analysis was used to detect the expression of emtrelated protein ncadherin ecadherin and mmp9 the solvent group was used as a baseline for the migration and invasion of cells three independent repetitions for each experiment were performed p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 emt epithelialmesenchymal transition mmp matrix metalloproteinase that of the 5fu treatment alone group in addition the cell viability of sw620 and lovo cells gradually decreased with the increasing dose of 5fu however after treatment with the combination of mmoll rg3 and µmoll fu for h the cell viability of sw620 cells was only which was not conducive to subsequent protein detection experiments thus mmoll rg3 and µmoll 5fu were chosen for subsequent experimentationcell clone formation assays were also used to detect in vitro proliferation of colon cancer cells as shown in fig 1c and d the number of colonies formed by the colon cancer cells treated with rg3 and fu was significantly lower than that of rg3 or fu alone these findings indicated that combined treatment of rg3 and 5fu enhanced the inhibition of colon cancer cell proliferation in vitrocombined treatment of rg3 and fu enhances the inhibition of cell migration and invasion the ability of tumor cells to invade and migrate is the key to tumor progression in the present study we compared the effects of different treatment conditions on the invasion and migration of colon cancer cells it was demonstrated that the invasion and migration ability of the colon cancer cells treated with rg3 combined with fu was significantly lower than that of rg3 or 5fu alone fig 2a and b epithelialmesenchymal transition emt is the source of tumor cell ability to acquire higher invasion and migration capacity thus we determined the levels of three emtrelated proteins ncadherin ecadherin and mmp9 and found that the expression of ncadherin and mmp9 protein in the rg35fu group was significantly lower than that of rg3 or fu alone group but 0concology reports figure effect of the combined treatment of rg3 and 5fu on the apoptosis of colon cancer cells in vitro a the percentage of apoptotic sw620 and lovo cells in the different groups b apoptosisrelated proteins [cleaved clcaspase clcaspase and apaf1] were assessed by western blot analysis in sw620 and lovo cells three independent repetitions for each experiment were carried out p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 apaf1 apoptotic protease activating factor the expression of ecadherin protein was significantly higher fig 2ccombined treatment of rg3 and fu promotes apoptosis of colon cancer cells fist we found that the apoptosis of the colon cancer cells treated with rg3 combined with 5fu was significantly higher than that of rg3 or fu alone fig 3a the levels of apoptosisrelated proteins in the sw620 and lovo cells were assessed by western blot analysis as shown in fig 3b expression levels of apaf1 cleaved clcaspase and clcaspase protein in colon cancer cells sw620 and lovo treated with rg3 were significantly increased and the expression of these apoptosisrelated protein in colon cancer cells following fu treatment was significantly higher than that treated with rg3 more importantly expression levels of these apoptosisrelated proteins in colon cancer cells treated with the combination of rg2 and 5fu were significantly higher than levels treated with rg3 or 5fu alonewe analyzed the cell cycle distribution of the colon cancer cells after treatment with the different agents as shown in fig 4a the percentages of colon cancer cells in the g0g1 phase treated with the rg3 and 5fu combination were significantly higher than the percentages following rg3 or 5fu alone similarly we also detected cell cycleassociated protein by western blot analysis as shown in fig 4b the expression levels of cyclin d1 cdk2 and cdk4 protein in colon cancer cells which were treated with the rg3 and 5fu combination were significantly lower than levels following treatment with rg3 or 5fu alonecombined treatment of rg3 and fu suppresses pi3kakt signaling in colon cancer cells the pi3kakt signaling 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on cell cycle progression of colon cancer cells in vitro a flow cytometry was used to analysis the cell cycle in colon cancer cells after treatment with rg3 mmoll or 5fu µmoll or the combination b cell cycleassociated protein cyclin d1 cdk2 and cdk4 were assessed by western blot analysis three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 cdk cyclindependent kinase pathway is a signaling pathway involved in cancer cell proliferation invasion and migration and its abnormal activation can confer high proliferation invasion and migration ability of cancer cells in the present study we found that the expression levels of pp85 p110 ppdk1 and pakt protein in the colon cancer cells which was treated with rg3 and 5fu combination were significantly lower than levels in the cells treated with rg3 or 5fu alone fig these results indicated that the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt signaling pathway in colon cancer cells in vitrocombined treatment of rg3 and fu suppresses tumor growth in nude mice based on the results of in vitro studies we further investigated the effects of the rg3 and 5fu combination on colon cancer cell proliferation and protein expression in nude mice sw620 cells were injected into the armpits of nude mice after weeks of treatment the mice were sacrificed and the weight and volume of tumor tissues were measured it was found that the weight and volume of tumor tissues in the rg35fu group were significantly lower than these parameters in the groups treated with rg3 or 5fu alone fig 6a and bmoreover western blot analysis was used to detect the expression of emtrelated proteins cell cyclerelated proteins and key proteins in the pi3kakt signaling pathway it was found that although the effects of the rg3 and 5fu combination were not as obvious as the in vitro results compared with rg3 of 5fu alone the overall trend in protein expression was consistent fig 6ce these results demonstrated that rg3 0concology reports figure effect of the combined treatment of rg3 and 5fu on pi3kakt signaling in colon cancer cells in vitro a and b western blot analysis was used to detect the expression of key proteins in the pi3kakt signaling pathway after treatment with rg3 mmoll or 5fu µmoll or the combination three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude micediscussionthe anticancer effect of 5fluorouracil 5fu is exerted mainly by interfering with tumor cell dna replication and it is a commonly used antitumor agent for the treatment of advanced colon cancer however since 5fu displays nonspecific cytotoxicity it also causes damage to normal cells causing irreversible renal dysfunction and severe gastrointestinal reactions these adverse effects limit its clinical application and further improvements in the efficacy of chemotherapy are needed therefore it is urgent to discover a drug that can enhance the chemotherapeutic effects of 5fu and reduce the 5fu toxicity when used in combination with 5fuginsenoside rg3 rg3 is one of the main active ingredients extracted from ginseng research has shown that ginsenoside rg3 has certain inhibitory effects on lung cancer breast and prostate cancer the antitumor mechanism of rg3 was that rg3 reduced the neovascularization probability of tumor recurrence proliferation and metastasis in tumors by inhibiting kdrvegf protein expression and blocking hif1αcox2vegf pathway in the present study we found that the combined treatment of rg3 and 5fu promoted the inhibition of colon cancer cell proliferation in vivo and in vitro tumor growth development and metastasis are closely related to cell proliferation the previous study found that rg3 inhibits the proliferation of tumor cells such as rg3induced egfrmapk pathway deactivation was found to inhibit melanoma cell proliferation by decreasing fut4ley expression rg3 was found to inhibit the proliferation of multiple myeloma cells by inducing the secretion of igf1 promoting tumor cell apoptosis is also a method of inhibiting tumor cell proliferation in the present study we found that the combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway in the mitochondrial pathway of apoptosis apoptosisrelated signals release cytochrome c by stimulating the mitochondrial outer membrane cytochrome c enters the cytoplasm which activates caspase9 by binding with apaf1 activation of caspase9 further activates caspase3 while the activated caspase3 can activate caspase leading to apoptosis in addition we also found that the rg3 and 5fu combination enhanced the number of g0g1 phase colon cancer cells and decreased expression of cyclin d1 cdk2 and cdk4 the cell cycle refers to the whole process that the cell undergoes from the completion of one division to the end of the next division and 0chong effects of ginsenoside rg3 on colon cancerfigure effects of the combined treatment of rg3 and 5fu on tumor growth and protein expression of colon cancer cells in vivo after weeks of treatment the mice were sacrificed tumor tissues were excised and the weight a and volume b of tumor tissues were measured ce total protein was extracted from the colon cancer tumor tissues and the expression of proteins was detected by western blot analysis five nude mice in each group and at least tumor tissues were used to evaluate protein expression p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 the regulation of the cell cycle is mainly achieved by the retention of the g1 phase when a cell is in the g1 phase there is an important node regulating the cell cycle the r point when the cell cycle is before the r point the cell needs the external growth factor to achieve the normal operation of the cell cycle after the cell cycle crosses the r point the cell cycle becomes a process that is controlled autonomously by the cell and no longer depends on the presence of external cytokines cyclin d1 is a g1sspecific cyclin and its main function is to promote the cell cycle from g1 to s by binding and activating the cyclindependent kinase cdk24 a unique cyclindependent kinase of g1 so as to promote cell proliferation invasion and migration of tumor cells are the most important features of malignant tumors and the important causes of death in patients with malignant tumors ncadherin ecadherin and mmp9 are three proteins that play important roles in cell epithelialmesenchymal transition emt whereas emt provides cells the ability to transfer and invade promoting tumor cell emt can inhibit the expression of intercellular junction protein resulting in decreased intercellular connectivity which is beneficial to the invasion and migration of tumor cells to surrounding healthy tissues previous studies have found that rg3 not only inhibits metastasis and invasion of lung cancer cells by inhibiting emt induced by transforming factor 1 but also inhibited the metastasis of prostate pc3m cells by downregulating the expression of aqp1 by downregulating mmp rg3 affected the metastasis and invasion ability of melanoma cells the present study demonstrated that the combined treatment of rg3 and fu significantly suppressed the invasion and migration ability of human colon cancer cell in vitro by altering emtrelated proteinfurthermore we also found that rg3 and 5fu combination inhibited the conduction of the pi3kakt signaling pathway in vivo and in vitro many studies have shown that the occurrence and development of tumors are the result of multifactor multigene and multipathway processes and the cell signal transduction pathway is crucial in the process of tumor development invasion and metastasis the phosphatidylinositol 3kinaseserinethreonine kinase b pi3kakt signaling pathway plays an important role in the regulation of solid tumors [eg liver cancer breast cancer colon cancer gastric cancer neuroblastoma ] and blood tumors [eg leukemia ] pi3k acts as a bridge molecule for the relationship between extracellular signals and cellular responses under the influence of a series of upstream or bypass signaling molecules it acts on the downstream of the effects of a variety of molecules thus promotes cell migration 0concology reports inhibits cell apoptosis accelerates the process of the cell cycle and promotes cell proliferation many previous studies have shown that traditional chinese medicine or traditional chinese medicine monomers can play an antitumor role by inhibiting the pi3kakt signaling pathway in conclusion rg3 enhances 5fu inhibiting proliferation invasion and migration of colorectal cancer cells and helps 5fu block g1 phase induced apoptosis in more colorectal cells all in all our study found that rg3 enhanced the anticancer effect of 5fu on colon cancer cell via pi3kakt pathwayacknowledgementsnot applicablefundingno funding was receivedavailability of data and materialsthe datasets used during the present study are available from the corresponding author upon reasonable requestauthors' contributionsxc made substantial contributions to the conception and design of the study and critically revised it for important intellectual content sh contributed to the acquisition of the data wc zh yw xm yh and zl analyzed and interpreted the data all authors read and approved the final manuscriptethics approval and consent to participateall animal and cell experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinapatient consent for publicationnot applicablecompeting intereststhe authors state that they have no competing interestsreferences siegel rl ward em and jemal a trends in colorectal cancer incidence rates in the united states by tumor location 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one e98921 gao y xiao x zhang c yu w guo w zhang z li z feng x hao j zhang k melatonin synergizes the chemotherapeutic effect of fluorouracil in colon cancer by suppressing pi3kakt and nfbinos signaling pathways j pineal res doi 101111jpi12380 wang sf wu my cai cz li m and lu jh autophagy modulators from traditional chinese medicine mechanisms and therapeutic potentials for cancer and neurodegenerative diseases j ethnopharmacol ernst e traditional chinese medicine for cancer br j cancer sun hy lee jh han ys yoon ym yun cw kim jh song ys and lee sh pivotal roles of ginsenoside rg3 in tumor apoptosis through regulation of reactive oxygen species anticancer res tang yc zhang y zhou j zhi q wu my gong fr shen m liu l tao m shen b ginsenoside rg3 targets cancer stem cells and tumor angiogenesis to inhibit colorectal cancer progression in vivo int j oncol wang j tian l khan mn zhang l chen q zhao y yan q fu l and liu j ginsenoside rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of nfκb mediated epithelialmesenchymal transition and sternness cancer lett joo e ha yw and kim ys abstract lb23 molecular mechanisms of ginsenoside rg3 related to apoptosis in human lung and pancreatic adenocarcinomas cancer res lb23 kim bj nah sy jeon jh so i and kim sj transient receptor potential melastatin channels are involved in ginsenoside rg3induced apoptosis in gastric cancer cells basic clin pharmacol kim sm lee sy cho js son sm choi ss yun yp yoo hs yoon dy oh kw han sb and hong jt combination of ginsenoside rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of nfkappa b eur j pharmacol yuan hd quan hy zhang y kim sh and chung sh 20sginsenoside rg3induced apoptosis in ht29 colon cancer cells is associated with ampk signaling pathway mol med rep liu tg huang y cui dd huang xb mao sh ji ll song hb and yi c inhibitory effect of ginsenoside rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice bmc cancer sun my ye y xiao l duan xy zhang ym and zhang h anticancer effects of ginsenoside rg3 review int j mol med longley db harkin dp and johnston pg fluorouracil mechanisms of action and clinical strategies nat rev cancer hokmabady l raissi h and khanmohammadi a interactions of the fluorouracil anticancer drug with dna pyrimidine bases a detailed computational approach struct chem rateesh s luis sa luis cr hughes b and nicolae m myocardial infarction secondary to fluorouracil not an absolute contraindication to rechallenge int j cardiol e331e333 shan x aziz f tian ll wang xq yan q and liu jw ginsenoside rg3induced egfrmapk pathway deactivation inhibits melanoma cell proliferation by decreasing fut4ley expression int j oncol luo y zhang p zeng hq lou sf and wang dx ginsenoside rg3 induces apoptosis in human multiple myeloma cells via the activation of bcl2associated x protein mol med rep cardone mh roy n stennicke hr salvesen gs franke tf stanbridge e frisch s and reed jc regulation of cell death protease caspase by phosphorylation science 0chong effects of ginsenoside rg3 on colon can | 0 |
"As one of the DNA repair genes ataxia-telangiectasia mutated (ATM) gene which is responsible for the multisystem autoxomal recessive disorder ataxia-telangiectasia (AT) plays a crucial role in the recognition signaling and repair of DNA damage especially DNA double-strand breaks (DSBs) [4] [5]. The ATM protein is a member of phosphoinositide 3-kinase (PI-3 kinases) and can be activated by DSBs caused by ionizing radiation or reactive oxygen intermediates [6] [7]. Once activated ATM can phosphorylate various downstream substates that function in cell cycle arrest apoptosis and DNA repair such as p53 NBS1 BRCA1 and Chk2 [8] [9]. Therefore genetic variants in ATM gene may lead to the structure and function change of the protein and act as important factors indicating individual susceptibility to cancer. ATM -111G>A (rs189037) resides in the promoter of ATM gene. Increasing studies have shown that variations in the DNA promoter sequence may potentially alter the affinities of multiple regulatory proteins-DNA interactions or the specificity of the transcriptional process [10][13]. Although this polymorphism makes no amino acid change the alleles may have different binding affinity to the transcription factor and exhibit different levels of mRNA expression [14] [15]. Zhang et al. [16]declared that ATM rs189037 AA genotype was associated with a lower ATM mRNA levels than GG genotype in lung tissue samples. Their results showed that the G-to-A change might create a transcriptional inhibitor-binding site for ATM rs189037 A allele promoter and subsequently reduce the ATM mRNA expression. Consequently lower expression of ATM might cause elevated sensitivity to ionizing radiation defects in the activation of cell cycle checkpoints a reduced capacity for DNA repair and abnormal apoptosis. All of these features would contribute to increased individual cancer susceptibility. In recent years a number of studies have evaluated the association between this polymorphism and cancer risk such as thyroid carcinoma [17] oral cancer [18] breast cancer [19] leukemia [20] nasopharyngeal carcinoma [21] glioma [22] and lung caner [23][25]. Previous studies of ATM rs189037 have included cigarette smokers as cases and controls that made it difficult to judge whether this polymorphism were associated with lung cancer or tobacco use. Considering the facts in China the incidence and death rate of lung cancer in women continues to increase and this phenomenon is frequently occurring in those who have never smoked. In order to have a better control of confounding of gender or smoking we performed a case-control study to identify the association between the polymorphism of ATM rs189037 and the risk of lung cancer in the non-smoking females in Chinese Han population. We also investigated the interaction between genetic polymorphism and environmental exposure in lung cancer. Methods Subjects This hospital-based case-control study included 487 lung cancer patients and 516 cancer-free hospital controls. All subjects were female non-smokers and they were from unrelated ethic Han Chinese. The cases were recruited during January 2002 to November 2012 at Liaoning Cancer Hospital & Institute. All patients were histologically confirmed to have lung cancer before any radiotherapy and chemotherapy. During the same time controls were selected from patients with other lung diseases but free of cancer history and symptom. Controls suffered mainly from bronchitis pneumonias fibrosis sarcoidosis chronic obstructive pulmonary disease and emphysema. Controls were all non-smoking females and frequency-matched to case subjects for age (±5 years). This study was approved by the institutional review board of China Medical University and written informed consent was obtained from each participant or each participant's representatives if direct consent could not be obtained. Data Collection A total of 10 ml of venous blood was collected from each patient. Patients were interviewed to collect information for demographics and environmental exposure at the time they were admitted to hospital. Information concerning demographic characteristics passive smoking cooking oil fume exposure fuel smoke exposure family history of cancer occupational exposure and dietary habit was obtained for each case and control by trained interviewers. An individual was defined as a smoker if she had consumed a total of 100 cigarettes in her lifetime; otherwise she was considered as a non-smoker. About fuel smoke exposure participants who used coal-fuel-burning stoves without chimneys were regarded as fuel smoke exposure. For exposure to cooking oil fumes participants were mainly asked about the method of cooking and eyes or throat irritation. For cooking methods participants were asked whether they cooked food in a stir-frying way and how many times a week; for eyes or throat irritation participants were asked how often they felt eyes or throat irritated by the oily smoke. There were four possible responses ranging from never seldom sometimes and frequently. Subjects were considered as cooking oil fume exposure if they met criteria as follows: (1) have cooked for over 15 years; (2) cooked food in a stir-frying way for more than twice a week; (3) felt eyes or throat irritated by oily smoke. Exposure for cooking oil fume was categorized as an indicator variable equal to 1 if participants reported frequently or sometimes and equal to 0 otherwise. Genotype Analysis Genomic DNA was extracted from peripheral blood samples by the conventional phenol-chloroform extraction method. SNP was genotyped by investigators blinded to case-control status in order to avoid any genotyping bias using TaqMan methodology and read with the Sequence Detection Software on an Applied Biosystems 7500 FAST Real-Time PCR System according to the manufacturer's instructions (Applied Biosystems Foster City CA). Amplification was done under the following conditions: 95°C for 10 min followed by 47 cycles of 92°C for 30 s and 60°C for 1 min. In this study 487 lung cancer patients and 516 controls were all genotyped successfully and 5% duplicated samples were randomly selected to assess the reproducibility for quality control with a concordance rate of 100%. Statistical Analysis The x2 test and t test were applied to estimate differences in demographic variables and distributions of genotypes between cases and controls. The association of genotypes of ATM rs189037 with risk of lung cancer was estimated by computing the odds ratios (ORs) and 95% confidence intervals (CIs) in unconditional logistic regression analysis. The Hardy-Weinberg equilibrium (HWE) was tested using goodness-fit x2 test to compare the genotype frequencies in the control subjects from those expected. A logistic regression model was used to evaluate gene-environment interactions. All data were analyzed with Statistical Product and Service Solutions (SPSS) v13.0 for Windows if not otherwise specified. All statistical analysis were two-sided and the significance level was set at P<0.05. Results Population characteristics A total of 487 lung cancer and 516 age-matched cancer-free controls were enrolled in this study. As shown in the mean ages of cases and controls (mean ±S.D.) were almost identical (56.5±11.7 and 56.3±12.5 respectively). All cases were female non-smoking lung cancer patients. No statistically significant difference was found between cases and controls in terms of age (P?=?0.248) and monthly income (P?=?0.084). Cases included 434 non-small cell lung cancer (NSCLC) patients and 53 small cell carcinoma patients. In the NSCLC cases there were 320 adenocarcinomas 73 squamous cell carcinomas and 41 other tumors with a variety of different pathologies (such as large cell carcinomas mixed cell carcinomas or undifferentiated carcinomas). .0096911.t001 Characteristics of lung cancer cases and controls. Variables Cases(%) Controls(%) P value Female 487 516 Mean age (years) 56.5±11.7 56.3±12.5 0.248a Income (yuan/month) 628.9±419.3 563.5±387.6 0.084a Never smoker 487 516 Histological type NSCLC 434(89.1) Adenocarcinoma 320(65.7) Squamous cell carcinoma 73(15.0) Small cell carcinoma 53(10.9) Other 41(8.4) a Student's t-test was used to compare the frequency distributions of demographic variables between the cases and controls. Association analysis The observed genotype frequencies among the control subjects was in agreement with that expected under the Hardy-Weinberg equilibrium (P?=?0.119). The distribution of ATM rs189037 genotypes among subjects were displayed in Table 2. Using subjects with the ATM rs189037 GG genotype as the reference group we calculated the ORs and 95%CIs for heterozygous carriers of GA genotype and homozygous carriers of AA genotype. No significant difference was observed between lung cancer cases and controls in each test (P>0.05). In order to increase the statistical power we combined the GA genotype with the AA genotype to compare with GG genotype as a dominant model and combined the GA genotype with the GG genotype to compare with AA genotype as a recessive model. The results indicated that individuals with AA genotype had a significantly elevated risk of lung adenocarcinoma compared with those carrying the GG or GA genotype (OR?=?1.44 95%CI 1.022.02 P?=?0.039). .0096911.t002 Table 2 Distribution of ATM rs189037 genotypes and ORs for lung cancer cases and controls. Genotype Cases(%) Controls(%) ORc 95%CI P overall (n?=?487) GG 148(30.4) 152(29.5) ref GA 240(49.3) 272(52.7) 0.91 0.681.20 0.494 AA 99(20.3) 92(17.8) 1.11 0.771.59 0.590 dominant modela 0.96 0.731.25 0.742 recessive modelb 1.18 0.861.61 0.313 NSCLC (n?=?434) GG 129(29.7) 152(29.5) ref GA 213(49.1) 272(52.7) 0.92 0.681.24 0.573 AA 92(21.2) 92(17.8) 1.18 0.811.71 0.397 dominant model 0.98 0.741.30 0.906 recessive model 1.24 0.901.71 0.192 Adenocarcinoma (n?=?320) GG 94(29.4) 152(29.5) ref GA 150(46.9) 272(52.7) 0.89 0.641.23 0.485 AA 76(23.7) 92(17.8) 1.33 0.901.99 0.156 dominant model 1.00 0.741.36 0.987 recessive model 1.44 1.022.02 0.039* Squamous cell carcinoma (n?=?73) GG 24(32.9) 152(29.5) ref GA 39(53.4) 272(52.7) 0.90 0.521.56 0.706 AA 10(13.7) 92(17.8) 0.69 0.321.51 0.355 dominant model 0.85 0.501.43 0.537 recessive model 0.74 0.371.50 0.400 *P<0.05. a GA+AA vs GG. b AA vs GA+GG. c adjusted for age and data were calculated by unconditional logistic regression. According to the results above we assumed that ATM rs189037 AA genotype might affect lung adenocarcinoma risk among non-smoking Chinese females. To test this hypothesis and explore the gene-environment interaction we adopted all the lung adenocarcinoma patients and cancer-free controls whose information about environmental risk factors were completely obtained such as fuel smoke exposure cooking oil fume exposure passive smoking and family history of cancer. Cases and controls were not included in the association analysis if any item of their environmental risk factors data was incomplete. After screening we had 242 lung adenocarcinoma cases and 277 cancer-free controls that were eligible. Selected demographic variables and environmental risk factors for the cases and controls were listed in Table 3." | 1 |
"purpose squamous cell carcinomas and adenocarcinomas are the most common types of cervical cancercompared to squamous cell carcinomas adenocarcinomas are more common in younger women and have apoorer prognosis yet so far no useful biomarkers have been developed for these two types of cancer in thefollowing study we examined the combination of cytokeratin p63 p40 and muc5ac for distinguishingsquamous cell carcinoma scc from adenocarcinoma of the cervix aecmaterials and methods a total of scc and aec were collected immunohistochemical analyses wereconducted to determine the expression of ck56 p63 p40 ck7 and muc5ac one pathologist who was blinded tothe patients clinical and pathological data interpreted the staining resultsresults muc5ac and ck7 were detected in and of aec cases compared to and of scccases p the specificity of muc5ac was higher than that of ck7 in aec p the sensitivity of muc5accombined with p40 or p63 was similar to that of ck7 but the specificity was slightly higher than that of ck7 inaec moreover the expression of muc5ac was correlated with the degree of tumor differentiation inadenocarcinomas p and was not related to the prognosis of cervical adenocarcinoma and subtypess muc5ac may be useful as a biomarker for differential diagnoses between squamous carcinoma andadenocarcinoma of the cervixkeywords cervical adenocarcinoma cervical squamous cell carcinoma muc5ac ck7 correspondence xiaofangzhangsdueducn hailing li and xiaotong jing contributed equally to this work2department of pathology school of basic medical science shandonguniversity jinan shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cli diagnostic pathology page of thelastintroductioncervical cancer is the fourth most common carcinomain women responsible for of cancerrelateddeaths worldwide [ ] squamous carcinoma is themost common type of cervical carcinoma followed byadenocarcinoma nevertheless overthreedecades a significant increase in adenocarcinoma caseshas been observed in many developed countries especiallyin younger women papsmear screening also knownas pap test is still considered the main screening methodfor cervical cancer especially for squamous carcinoma compared to squamous carcinoma the adenocarcinomaof the cervix is more common in younger women and hasa poorer prognosis therapeutic approaches includechemoradiotherapy ccrt which has been proven tobe effective for squamous carcinoma of the cervix but notfor adenocarcinoma of the cervix due to its highchemo and radioresistance therefore differentiatingadenocarcinoma from squamous carcinoma is importantin order to provide patients with most suitable therapyp63 p40 and cytokeratin 56ck56 are the mostcommon panel ofimmunochemical markers for thediagnosis of squamous carcinoma p63 and ck56are traditional markers that indicate squamous differentiation in primary lung neoplasms most squamouscarcinomas and large cell carcinomas are positive forck56 warth found that the probability of acorrect sqcc diagnosis using ck56 is p63 a transcriptional regulator has a crucial role in thedevelopment and differentiation of stratified squamousepithelium it is usually strongly expressed in the basalkeratinocytes [] vosmik analyzed patientswith cervical squamous cell carcinoma and found that had positive expression of p63 p40 is a new specific marker for distinguishing squamouscarcinomas from adenocarcinoma whose specificity isabout in lung carcinomas however the positiveexpression of ck56 p63 and p40 are only found in a fewadenocarcinomas [ ] kriegsmann suggested theuse of either ck56 or p40 over p63 in the routine diagnostic setting ck7 is expressed in many ductal andglandular epithelial cells mainly gallbladder hepatic ductsand pancreatic ducts in tissues of the female genital tractovary endometrium fallopian tube and cervix and in thebreast lung and urinary tract tissues in the normalcervical tissue and adenocarcinoma ck7 staining wasobserved in the columnar cells of endocervical glandshashiguchi found the different rates of ck7 in patientswith cervical intraepithelial neoplasia and those with invasivecarcinomas vs [ ] thus far no efficientmarkers have been developed for distinguishing squamouscell carcinoma and adenocarcinoma in the endocervixmucins are a family of large glycoproteins expressedon the epithelial cell surfaces including ducts of lacrimalglands in the eye salivary glands the lining of the respiratory gastrointestinal urothelial and reproductive tracts muc5ac belongs to gelforming mucins multiple histological studies have highlighted that muc5acis expressed in the conjunctiva middle ear nasopharynxlungs gallbladder and stomach under normal conditionswhere it provides protection to corresponding epithelialsurfaces from different factors some research hasshown that muc5ac may be a potential biomarker inpancreatic cancer tissues dimaio found thatanterior gradient homolog and muc5ac are usefulpositive markers of adenocarcinoma in the setting ofabsent or diminished p63 and cytokeratin staining inesophageal carcinoma it is also expressed in theendocervix yamanoi found that muc5ac waslargely expressed in typical legh atypical legh gasmda and gasnonmda thus we speculated thatmuc5ac could be expressed in other adenocarcinomasand might be used for the differential diagnosis of adenocarcinoma and squamous carcinoma the aim of thisstudy was to examine the combination of cytokeratin p63 p40 and muc5ac for distinguishing squamous cellcarcinoma scc from the adenocarcinoma in the cervixaecmaterials and methodstissue sampleswe analyzed poorly to moderately differentiated cervical squamous carcinoma scc and adenocarcinomasof endocervix aec all tissues were collected from thedepartment of human pathology of qilu hospitalshandong university china from to specimenswere retrieved from the pathology files of the departmentof pathology at the same hospital after collection all specimens were fixed in buffered formalin hematoxylin eosin he stains were available for review paraffin blockswere used for immunohistochemical staining all the slideswere reviewed by two experienced pathologistshistopathological and clinical variables including agetumor size differentiationinfiltrate depth and lymphnode metastasis were summarized in table followupinformation was available in aec with the followuptime ranging from to months mean monthsimmunohistochemistryfour to five micronthick paraffin sections of the cases were dewaxed rehydrated in graded alcohols andprocessed using the pv9000 detection kit zsbio commerce store beijing china briefly antigen retrieval wasperformed in a microwave oven for min in mm trisedta buffer mm tris base mm edta solution tween ph endogenous peroxidase activitywas blocked with a h2o2methanol solution for min slides were then incubated in normal goat 0cli diagnostic pathology page of table comparison of clinicopathological features between cervical squamous cell carcinoma and cervical adenocarcinomasquamous cell carcinomasn adenocarcinoman Ï p valueage¤ size cm ¥ unknowndifferentiationpoormoderatewellunknown infiltrate depth of mesenchyme¤ unknownlymph node metastasisnoyesunknown serum for min to prevent nonspecific binding sampleswere then incubated overnight at °c with a primary antibody phosphate buffered saline pbs was used instead ofthe first antibody as a negative control consequentlysamples were incubated with reagent atroomtemperatureroomfor min and reagent attemperature for min finally the tissues were stainedwith diaminobenzidine dab the antibodies used in thisstudy are listed in table scoring methodstaining results were interpreted by one pathologist whowas blinded to the patients clinical and pathologicaldata for ck56 ck7 and muc5ac more than oftumor cells with a membrane or cytoplasmic brownyellow granules were considered positive for p63 andp40 the positive standard was that more than oftumor cells have brownyellow granules in the nucleusstatistical analysisstatistical analysis was performed with spss softwareversion spss inc chicago ii usa chisquareor fishers exacttests were used when comparingfrequencies between two groups probability values lessthan were considered statistically significantresultsthe expression of ck56 p63 p40 ck7 and muc5ac inscc and aecihc for the five proteins was performed on humanprimary cervical cancersincluding scc and aec as shown in fig and fig muc5ac ck56and ck7 were mainly expressed in the cell membranetable immunohistochemical antibodiesantibodymuc5acnozm0395ck56ck7p40p63zm0313zm0071zm0472zm0406vendorzsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinazsbio commerce store beijing chinadiluationready to useready to useready to useready to useready to use 0cli diagnostic pathology page of fig the expression of ck56 p63 p40 ck7 and muc5ac in a case of poordifferentiated squamous cell carcinoma by ihc a he b ck56positive staining c p63 positive staining d p40 positive staining e ck7 positive staining f muc5ac negative staining Ãfig the expression of ck56 p63 p40 ck7 and muc5ac in case of poordifferentiated adenocarcinoma invasive stratified mucinproducingcarcinoma ismile by ihc a he b ck56 negative staining c p63 negative staining d p40 negative staining e ck7 negative staining f muc5acpositive staining à 0cli diagnostic pathology page of and cytoplasm while p40 and p63 were mainly locatedin the nucleussignificant effect on the prognosis of cervical adenocarcinoma patients p as shown in fig tumorin thecells ofwe found that muc5ac exhibited prominent immucervical aecnoreactivitymuc5ac and ck7 were detected in and of aec cases compared to and of scc casesbesides for aec the specificity of muc5ac was muchhigher than that of ck7 p moreover the sensitivity of ck56 p40 and p63 was and respectively and the specificity was and respectively in aec table through the combined detection of p40 or p63 wecompared muc5ac and ck7 again we found that thesensitivity and specificity of muc5ac in aec combinedwith p40 or p63 were and respectively and respectively the sensitivity and specificity of ck7 combined with p40 or p63 were and and respectively table thesensitivity of muc5ac combined with p40 or p63 wassimilar to that of ck7 while the specificity was slightlyhigher than that of ck7expression of muc5ac and ck7 in cervicaladenocarcinoma subtypeswe further detected the expression of muc5ac insubtypes of aec table among cases of usualtype cervical adenocarcinoma cases were muc5acpositive and cases were ck7 positive and there wasno statistical difference p in cases of mucinous adenocarcinoma nosthe expression rate ofmuc5ac and ck7 were both moreover out of cases of gastric mucinous adenocarcinomaexpressed muc5ac and of them were ck7 positivep the positive rate of the muc5ac in mucinouscarcinoma intestinal type villous tubular adenocarcinomaendometrioid adenocarcinoma clear cell carcinoma serouscarcinoma adenosquamous carcinoma and invasive stratified mucinproducing carcinoma ismile was and respectively the expressionrate of muc5ac had no statistical difference among thesesubtypes all p correlation between muc5ac expression andclinicopathological characteristics in cervicaladenocarcinomathis study further analyzed the relationship between theexpression of muc5ac and clinicopathological featuresin cervical adenocarcinoma table the expression oftumormuc5ac was correlated with the degree ofdifferentiation p a lower degree oftumordifferentiation was associated with a lower expressionrate of muc5ac there was no significant correlationbetween the expression of muc5ac protein and agetumor size depth of myometrialinvasion and lymphnode metastasis all p kaplan meier analysisrevealed that the expression of muc5ac protein had nodiscussion and sidentification of previously unutilized sensitive biomarkers is still a priority for improved differential diagnosis of cervical aec and scc at present ck56 p63p40 and ck7 are the main biomarkers for differentiatingcervical adenocarcinoma from squamous cell carcinomack56 is a kind of high molecular weight basal cellkeratin 58kda and 56kda which is mainly expressed inthe basal cells of squamous epithelium and ductal epithelium and some squamous epithelial germinal layercells myoepithelial cells and mesothelial cells butpoorly expressed in glandular epithelial cells someresearch results showed that ck56 has high sensitivityand specificity in the diagnosis ofsquamous celltable sensitivity and specificity of muc5acãck56ãck7ãp40ãp63 in cervical squamous cell carcinoma and adenocarcinomamarkerssensitivityspecificitysquamous cell carcinomasn adenocarcinoman muc5acck7ck56p40p63ck56and p40ck56and p63muc5acand p40muc5acand p63ck7and p40ck7and p63 0cli diagnostic pathology page of table the correlation of muc5ac and the clinical variants inthe cervical adenocarcinomathe expression of muc5acpositivenegativeÏ valuep valueage¤ v size cm ¥ differentiationpoorwellmoderateinfiltrate depthof mesenchyme¤ lymph nodemetastasisnoyescarcinoma [] in contrast other studies showedhigh sensitivity but low specificity when diagnosing thistype of tumor p63 is a member of the p53 family a classical tumorsuppressor gene family it is located on chromosome3q27 filho showed good sensitivity whendetecting squamous cell carcinoma with a positive rateof contrary kaufmann suggested thatp63 could also be expressed in a small number of adenocarcinoma basal cell carcinoma and transitional epithelial carcinoma moreover p63 can also be used as amarker of myoepithelial cells and prostate basal cellstherefore p63 lacks absolute specificity for squamousdifferentiationp40 is a subtype of p63 protein expressed in squamousepithelial cells including epidermis and hair folliclesurothelial cells myoepithelial cells ofthe mammarygland sweat gland and salivary gland and basal cells ofthe prostate which are highly specific in labeling squamous epithelium bishop showed that in cases of squamous cell carcinoma of the lung and cases of adenocarcinoma of the lung the sensitivity andspecificity of p63 were and respectivelythe sensitivity and specificity of p40 in the diagnosis ofsquamous cell carcinoma of the lung were and respectively therefore p40 is considered as ahighly specific and sensitive tumor biomarker of squamous epithelial origin in this study we used immunohistochemistry to detectck56 p63 and p40 in cervical squamous cell carcinomaand adenocarcinoma the sensitivity of ck56 p40 andp63 was and respectively and thespecificity was and respectivelymoreover the specificity of ck56 is slightly lower thanthat of p40 and p63 we also found that a combinationof ck56 with p40 or p63 slightly decreased the sensitivity and and increased the specificity and which in turn increased the accuracy of diagnosing squamous cell carcinomack7 is a kind of low molecular weight keratin mainlyexpressed in glandular epithelium and transitional epithelial cells of most normal tissues many studieshave found that ck7 is not only expressed in adenocarcinoma but also in squamous intraepithelial neoplasiacervical squamous cell carcinoma lung squamous cellcarcinoma and esophageal squamous cell carcinomalee found a positive expression of ck7 in fig survival analysis of muc5ac expression in cervical adenocarcinoma 0cli diagnostic pathology page of table expression of muc5ac and ck7 in differentadenocarcinoma subtypessubtypesmuc5acusual typeÏ valueck7p valuepositivenegativemucinous adenocarcinoma nospositivenegativegastric typepositivenegativeintestinal typepositivenegativevillous tubular adenocarcinomapositivenegativeendometrioid adenocarcinomapositivenegativeclear cell carcinomapositivenegativeserous carcinomapositivenegativeismilepositivenegativeadenosquamous carcinomapositivenegativeismile invasive stratified mucinproducing carcinoma cases with scc and cases withciniii furthermore yamada found that ck7expression in esophageal squamous cell carcinoma butalso in iiiaiib stage esophageal squamous cell carcinoma suggest poor tumor differentiation and thus canbe used as an independent prognostic factor ourstudy showed that the positive rate of ck7 was incervical poorly differentiated squamous cell carcinomawhich further suggested that ck7 is not an ideal markerfor differentiation between squamous cell carcinoma andadenocarcinomamucin is a high molecular weight glycosylated proteinsecreted by epithelial cells in the respiratory tractgastrointestinal tract and urogenital tract which has animportant role in the protection of epithelium cell adhesion signal transduction immune activation and inhibition at present at least mucins have been found inthe female reproductive system riethdorf and albarracin used immunohistochemistrymethods to detect the expression of muc5ac in different female reproductive system malignant tumors theyfound that muc5ac was highly expressed in cervicaladenocarcinoma and poorly expressed inendometrial adenocarcinoma all of themwere expressed in the primary ovarian mucinous tumor but not in colon adenocarcinoma therefore they concluded that muc5ac could beused as an effective marker to distinguish the origin ofpelvic tumors and distinguish primary ovarian tumorsand colorectal metastasis as well as endometrial adenocarcinoma from cervical metastasis [ ] in thisstudy we found positive expression of muc5ac in cases of cervical adenocarcinoma and in cases of squamous carcinoma which wasconsistent with riethdorfs study the sensitivity ofmuc5ac and ck7 to cervical adenocarcinoma was and respectively but the specificity ofmuc5ac was much higher than that of ck7 through the joint detection of p40 or p63 wecompared muc5ac and ck7 again and found that thesensitivity and specificity of muc5ac combined withp40 or p63 were and respectively and respectively the sensitivity and specificity ofck7 combined with p40 or p63 were and and respectively these results showed thatthe sensitivity of muc5ac combined with p40 or p63was similar to that of ck7 butthe specificity wasslightly higher than that of ck7 therefore muc5ac issuperior to ck7 in the diagnosis of cervical adenocarcinoma and squamous cell carcinomabesides we preliminarily detected the expression ofmuc5ac in different types of cervical adenocarcinomaand found no significant difference these data suggestedthat muc5ac has no diagnostic significance in the classification of cervical adenocarcinoma at the same time weanalyzed the relationship between the expression ofmuc5ac and the prognosis of cervical adenocarcinomaand the result revealed that muc5ac was not related tothe prognosis of cervical adenocarcinomaoverall our observations strongly suggest that muc5acmay be useful as a biomarker for differential diagnosesbetween squamous carcinoma and adenocarcinomaabbreviationsscc squamous cell carcinoma aec adenocarcinoma of the cervixck cytokeratin he hematoxylin eosin pbs phosphate buffered salinedab diaminobenzidine ismile invasive stratified mucinproducingcarcinoma 0cli diagnostic pathology page of authors contributionsxiaofang zhang designed the study and drafted the manuscript hailing liand xiaotong jing analyzed the data and carried out theimmunohistochemistry jie yu and tingguo zhang read the pathologicalsections jinan liu collected the clinical data and carried our followupshiming chen made the slides the authors read and approved the finalmanuscript downey p cummins r moran m gulmann c if it's not ck56 positive ttf negative it's not a squamous cell carcinoma of lung apmis warth a muley t herpel e meister m herth fj schirmacher p weichert whoffmann h schnabel pa largescale comparative analyses ofimmunomarkers for diagnostic subtyping of nonsmallcell lung cancerbiopsies histopathology fundingthis work was supported by the national natural science foundation ofchina no and technology development foundation of yantaino ws017availability of data and materialsnot applicableethics approval and consent to participateall tissue samples from patients were collected and protocols wereperformed according to the procedures approved by the research ethicscommittee of shandong medical university all patients provided informedconsentcompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology weifang traditional chinese hospital weifangshandong p r china 2department of pathology school of basic medicalscience shandong university jinan shandong p r china 3department ofpathology the fourth hospital of jinan the third affiliated hospital ofshandong first medical university jinan shandong p r china 4departmentof oncology yuhuangding hospital yantai shandong p r china5department of pathology school of basic medical science shandonguniversity jinan shandong p r chinareceived july accepted august referenceskurman rj carcangiu ml herrington cs who classification of tumours offemale reproductive ans4th ed lyon iarc press takeuchi s biology and treatment of cervical adenocarcinoma chin jcancer res young rh clement pb endocervical adenocarcinoma and its variants theirmorphology and differential diagnosis histopathology forouzanfar mh foreman kj delossantos am lozano r lopez ad murraycj naghavi m breast and cervical cancer in countries between and a systematic analysis lancet galic v herzog tj lewin sn neugut ai burke wm lu ys hershman dlwright jd prognostic significance of adenocarcinoma histology in womenwith cervical cancer gynecol oncol favero g pierobon j genta ml araujo mp miglino g del cpdm deandrade ch fukushima jt baracat ec carvalho jp laparoscopicextrafascial hysterectomy completion surgery after primary chemoradiationin patients with locally advanced cervical cancer technical aspects andoperative outcomes int j gynecol cancer rose pg java jj whitney cw stehman fb lanciano r thomas gm locallyadvanced adenocarcinoma and adenosquamous carcinomas of the cervixcompared to squamous cell carcinomas of the cervix in gynecologiconcology group trials of cisplatinbased chemoradiation gynecol oncol ma y fan m dai l kang x liu y sun y xiong h liang z yan w chen kexpression of p63 and ck56 in earlystage lung squamous cell carcinoma isnot only an early diagnostic indicator but also correlates with a goodprognosis thorac cancer kaufmann o fietze e mengs j dietel m value of p63 and cytokeratin as immunohistochemical markers for the differential diagnosis of poorlydifferentiated and undifferentiated carcinomas am j clin pathol barbieri ce pietenpol ja p63 and epithelial biology exp cell res senoo m pinto f crum cp mckeon f p63 is essential for the proliferativepotential of stem cells in stratified epithelia cell pozzi s zambelli f merico d pavesi g robert a maltere p gidrol xmantovani r vigano ma transcriptional network of p63 in humankeratinocytes plos one 200943e5008 vosmik m laco j sirak i beranek m hovorkova e vosmikova h drastikovam hodek m zoul z odrazka k prognostic significance of humanpapillomavirus hpv status and expression of selected markers her2neuegfr vegf cd34 p63 p53 and ki67mib1 on outcome after chemoradiotherapy in patients with squamous cell carcinoma of uterine cervixpathol oncol res nobre ar albergaria a schmitt f p40 a p63 isoform useful for lung cancerdiagnosis a review of the physiological and pathological role of p63 actacytol stolnicu s hoang l hankobauer o barsan i terinte c pesci a avielronens kiyokawa t alvaradocabrero i oliva e and others cervicaladenosquamous carcinoma detailed analysis of morphologyimmunohistochemical profile and clinical outcomes in cases modpathol toyoshima m momono y makino h kudo t oka n sakurada j suzuki hkodama h yoshinaga k cytokeratin 7positivecytokeratin 20negative cecaladenocarcinoma metastatic to the uterine cervix a case report world jsurg oncol hashiguchi m masuda m kai k nakao y kawaguchi a yokoyama maishima s decreased cytokeratin expression correlates with theprogression of cervical squamous cell carcinoma and poor patientoutcomes j obstet gynaecol res lee h lee h cho yk cytokeratin7 and cytokeratin19 expression in highgrade cervical intraepithelial neoplasm and squamous cell carcinoma andtheir possible association in cervical carcinogenesis diagn pathol krishn sr ganguly k kaur s batra sk ramifications of secreted mucinmuc5ac in malignant journey a holistic view carcinogenesis thornton dj rousseau k mcguckin ma structure and function ofthe polymeric mucins in airways mucus annu rev physiol rose mc voynow ja respiratory tract mucin genes and mucinglycoproteins in health and disease physiol rev balmaña m duran a gomes c llop e lópezmartos r ortiz mr barrabés sreis ca peracaula r analysis of sialyllewis x on muc5ac and muc1mucins in pancreatic cancer tissues int j biol macromol dimaio ma kwok s montgomery kd lowe aw pai rkimmunohistochemical panel for distinguishing esophageal adenocarcinomafrom squamous cell carcinoma a combination of p63 cytokeratin muc5ac and anterior gradient homolog allows optimal subtyping humpathol yamanoi k ishii k tsukamoto m asaka s nakayama j gastric gland mucinspecific oglycan expression decreases as tumor cells progress from lobularendocervical gland hyperplasia to cervical mucinous carcinoma gastrictype virchows arch reisfilho js simpson pt martins a preto a gartner f schmitt fcdistribution of p63 cytokeratins and cytokeratin in normal and neoplastic human tissue samples using tarp4 multitumor tissuemicroarray virchows arch yamada a sasaki h aoyagi k sano m fujii s daiko h nishimura myoshida t chiba t ochiai a expression of cytokeratin predicts survival instage iiiaiib squamous cell carcinoma of the esophagus oncol rep baker ac eltoum i curry ro stockard cr manne u grizzle we chhieng dmucinous expression in benign and neoplastic glandular lesions of theuterine cervix arch pathol lab med 0cli diagnostic pathology page of riethdorf l o'connell jt riethdorf s cviko a crum cp differentialexpression of muc2 and muc5ac in benign and malignant glandularlesions of the cervix uteri virchows arch albarracin ct jafri j montag ag hart j kuan sf differential expression ofmuc2 and muc5ac mucin genes in primary ovarian and metastatic coloniccarcinoma hum pathol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
" inhibiting suchtarget protein reduces the virulence of pathogens and reduces thetherapeutic resistance in the case of cancer we first come to the basicunderstanding of the role of grp78 in healthy and diseased cells grp78 in normal versus stressed cellunder normal conditions grp78 is found bound to three essentialenzymes that regulate cell growth differentiation apoptosis and signaling [] these enzymes are activating transcription factor atf6 protein kinase rnalike endoplasmic reticulum kinase perkand inositolrequiring enzyme ire1 which are inactivated throughbinding to grp78 in the er lumen under the pressure of unfoldedproteins in the er grp78 releases atf6 perk and ire1 and theenzymes are activated once activated the enzymes atf6 perk andire1 upregulate transcription of chaperones inhibit the translationand enhance protein folding endoplasmic reticulum assisted degradation erad and other function that have been reviewed byothers [] see also fig if the pressure of the unfolded proteins is not relieved the upr will direct part of the er to autophagyerphagy if it is not enough the whole cell will undergo apoptosis[]on the other hand under the stress of unfolded proteins grp78 canescape the er retention and translocates to the cytoplasm and on thecell membrane and become membraneexposed termed cellsurface corresponding authoremail addresses abdoscicuedueg aelfikyictpit aa elfiky101016jlfs2020118317received may received in revised form july accepted august available online august elsevier inc all rights reserved 0caa elfiky life sciences fig functional aspect of grp78 in normal versus stress condition in normal state left the grp78 is located in the lumen of the endoplasmic reticulum erbound to and inactivating atf6 blue triangle perk red circle and ire1 yellow square enzymes in the stress condition right the enzymes are free to do theirjobs atf6 is translocated to golgi apparatus to be cleaved then again translocated to the nucleus and helps in upregulating chaperones such as grp78 perk inhibitsthe translation and protein synthesis while ire1 enhances the folding and erad under the pressure of the unfolded proteins the grp78 escapes the er retention andtranslocate to the cytoplasm and the cell membrane csgrp78 is subjected to the recognition of pathogenic proteins spike and envelope viral protein and coatproteins of fungi for interpretation of the references to colour in this figure legend the reader is referred to the web version of this csgrp78 fig this csgrp78 characterizes many aggressive types of cancers such as breast ovarian pancreatic and coloncancers [] additionally csgrp78 was reported to facilitate pathogenic entry both viral and fungal infections zika viruszikv dengue virus denv hepatitis c virus hcv human papilloma virus hpv ebola virus ebov middleeast respiratorysyndrome coronavirus mers cov japanese encephalitis virus jevcoxsackievirus a9 and borna disease virus bdv are among viralinfections that reported grp78 association with viral proteins andgrp78 upregulation in infected cells [] additionally recentstudies hypothesized the association of csgrp78 with the spike protein of sarscov2 to help in virus attachment and host cell entry the primary binding viral protein to grp78 is spike proteins in coronaviruses and envelope proteins for the other viruses [] besidesthe spore coat protein homolog coth3 of rhizopus oryzae the causative fungus for mucormycosis is reported to bind to csgrp78 on endothelial cells and the binding is responsible for adherence and invasionof the fungus since the association of the viral or pathogen infection and elevatedlevels of csgrp78 expression researchers are focused on targetinggrp78 to prevent or even weaken the pathogenic infection reducingthe concentration of grp78 over the cell membrane would reduce thenumber of internalized pathogenic ps and hence reduce the infection additionally when we target csgrp78 the pathogen virulence would be diminished at the same time cancerassociated resistance would also be dimensioned which becomes of the highestpriorities in dual diseases such as viral or fungal infections in cancerpatients grp78 associated radio and chemoresistancechemoresistance is the resistance of a tumor to chemotherapy itwas an old observation while the mechanism of grp78inducedchemoresistance in cancer cells was not fully understood twomechanisms may be responsible for the chemoresistance the upr prosurvival branch and the receptormediated activation of the aktpi3kphosphoinositide 3kinase pathway alternatively the proapoptotic action of the upr could be compensated by the activation ofthe aktpi3k pathway resulting in cell survival the extracellular loopof cleft lip and palate transmembrane 1like clptm1l is essentialfor gemcitabine resistance and interaction with grp78 additionally natural products such as isoliquiritigenin a chalconetypeflavonoid were able to reduce the chemoresistance and colonyformingability of oral squamous cell carcinomas it is reported that theprior treatment of acidic stress protects the human dermal microvascular endothelial cells from apoptosis by reduced the cleavage ofcaspase which was supposed to be due to the presence of grp78 onthe membrane of er that suppress caspase activation nonsmallcell lung cancer nsclc and glioblastoma multiformegbm have a low survival rate the overexpressed grp78 on the cellsurface is the primary reason for the radioresistance in nsclc andgbm targeting cellsurface grp78 enhances the apoptosis andreduces cell proliferation colony formation and downregulates thecrucialintracellular phosphatidylinositol3kinaseprotein kinase bmammalian target of rapamycin pi3kaktmtor signaling essentialin the cell cycle growth and survival besides tumor growthis delayed with enhanced efficacy of the radiation treatment upon antigrp78 antibody administration in mice in breast cancer grp78 is overexpressed while the amount of cellsurface grp78 is increased upon the treatment with the antiangiogenicfactor combretastatin a4p additionally cancer cells treated withdoxorubicin showed less resistance when treated with grp78 neutralizing antibodies [] generally elevated levels of grp78 areindicative of cancer aggressivity targeting cancer cellsurface grp78 isa successful strategy to reduce the radioresistance and chemoresistance of tumors 0caa elfiky in this review the focus is not only on some of the previous trials touse antigrp78 to treat cancer but also in the diagnosis see the peptideinhibitors section below grp78 targeting strategiesdifferent strategies are used to reduce the burden of overexpressedcsgrp78 different compounds show binding affinity to csgrp78 once bound to a substrate the csgrp78 will be internalized tothe cell hence the concentration of the membranebound grp78 willbe reduced once the level of grp78 over the cell surface is droppedthe pathogens will not be able to enter the host cell through grp78hence the virulence will be reduced the inhibitory molecules that cantarget csgrp78 include phytochemicals peptides and antibodies andwill be discussed in detail in the next sections the inhibitors competewith the pathogen recognizing proteins such as spike envelope orcoat proteins for the csgrp78 substratebinding domain this domain of the grp78 is reported to be responsible for the binding ofgrp78 to unfolded proteins inside the lumen of the er through itshydrophobic batches phytochemicalsphytochemicals are compounds found in plants and have a varietyof effects on protein function they are derived from fruits vegetables beans grains and some other plants phytochemicalshave a protective role because their antioxidant characteristics whichplay a vital role in the protection of cells against oxidative damage anddecreasing the probability of cancer propagation via the reactiveoxygen species ros which can induce stress in er apoptosis initiated by the er if there is uncontrolled damage in cells wesummarize some phytochemicals crucial in cell stress relief throughinhibiting the master of upr grp78 galangin and 6shogaolgalangin is a flavonol produced from rhizomes of alpinia officinarum which belongs to the ginger family and grows in southeast asiagalangin works as a suppressor for cell proliferation in hepatocellularcarcinoma it raises er stress through the upregulation of the uprtarget genes cebp homologous protein chop grp78 glucoseregulating protein grp94 and cytosolic ca2 er is the primary site for intercellular calcium ions hence rising cytosolic ca2disrupts the function of er chaperones which induce er stress leadingto the activation of upr and subsequent upregulation of grp78 galangin upregulates er stress which inhibits tumor progressionthrough inducing apoptosis 6shogaol is produced by dehydration of 6gingerol and generatedfrom rhizomes of ginger when treating hepatocellular carcinomahcc cellline with 6shogaol cancer cells develop apoptotic phenotypes signs such as nuclear shrinkage and condensation in chromatin activation of chop expression and perk dephosphorylationinitiates reactions of caspase cascade which induce apoptosis in hccsignificant stimulation was observed in er stressrelated proteinswhich induce apoptosis by 6shogaol through rising in the upr expression grp94 grp78 and hsp70 studies proved that exposing cancer cells to 6shogaol and the activator of the perkeif2αpathway salubrinal together for a specific time induce er stress whichleads to cell apoptosis salubrinal alone enhances the phosphorylation of eif2α in the human hepatocarcinoma cell line smmc7721with negligible toxicity this reveals the significant therapeuticeffect of antigrp78 against malignancies fungisulphureuine b is produced from laetiporus sulphureus and tested byglioma cells to detect antiproliferative properties studies revealed thatsulphureuine b provides er stress by raising the level of expression oflife sciences chop caspase12 and grp78 which prevents separation of grp78from perk atf6 and ire1 which initiates upr additionallymushrooms contain pcoumaric acid and caffeic acid that proved itsbinding affinity against grp78 sbd in silico hence suggested to be apossible inhibitor for overexpressed grp78 in cancer cells or cell infected with viruses including sarscov2 grape seeds and skinproanthocyanidins and resveratrol extracted from grapeseedsexposing colorectal cancer cell crc to grape seed extract which has ahigh amount of proanthocyanidins and resveratrol leads to a modification in grp78 and protein disulfide isomerase pdi which have asignificant role in cell apoptosis which leads crc to undergo apoptoticpathway leading to inhibition of the targeted cell to proliferation on the other hand caffeic acid and pcoumaric acid polyphenolsfound in the grape skin have a protective role against photooxidativedamage additionally it has a preexposure protective role for thehuman retinal pigment epithelial cells arpe19 against blue lightassociated apoptosis in a dosedependent manner by promoting grp78expression in contrast grp78 knockdown inhibited this protective role as mentioned before caffeic acid and pcoumaric are suitablebinders to grp78 sbd in silico phytoestrogensestrogen receptorpositive breast cancer cells are responsive tohormonal therapy by blocking the estrogen synthesis leading to estrogenstarvation it was reported that grp78 plays a vital role inresist estrogenstarvation induced apoptosis in breast cancer cellshence it was suggested to dualtarget the grp78 during treating estrogenpositive breast cancer if the expression level of the grp78 ishigh to improve the efficacy and reduce the resistance it wasreported that grp78 interacts with estrogen due to the critical role ofgrp78 in folding the hormonebinding domain of estrogen receptors additionally grp78 targeting was suggested as a therapeuticstrategy to sensitize cancer cells to chemotherapy in endometrial cancerestrogen induced grp78 expression phytoestrogens are found incicer arietinum and include daidzein genistein formononetin andbiochanin a both estrogens estriol and estradiol and the fourphytoestrogens are found to be recognized by grp78 sbd and henceare suggested as possible grp78 inhibitors in silico it was concluded that estrogens and phytoestrogens are the best binders to thegrp78 while the binding affinities range from down to kcalmol this indicates an excellent binding affinity to grp78sbd even better than a selective cyclic peptide pep42 despiteits phytoestrogen activity genistein activates the apoptosis processthrough upr by upregulation of grp78 and cebp homologous proteinchop also termed growth arrest and dna damage gadd153and nuclear translation of gadd153 in hcc cells epigallocatechin3gallateepigallocatechin3gallate egcg is a polyphenol found in greentea and has an antiproliferative effect on breast cancer and melanoma besidesit has an inhibition effect against grp78 functionthrough direct interaction with the atp binding site of grp78 competing against atp binding egcg increases the therapeutic efficacy of temozolomide when exposed to glioblastoma cells in vivo byinhibition of grp78 olive leaf extract and honeybee hive propolisthe olive leaf extracts active ingredient hydroxytyrosol show goodbinding affinity to the grp78 sbd in silico hydroxytyrosolproved its role as a prophylactic agent against myocardial infarctionmediated apoptosis caffeic acid phenethyl ester cape is foundin the hive propolis of the honeybee cape shows in silico binding affinity against grp78 sbd that is comparable to that of the cyclicpep42 a selective grp78 peptide besides cape induces er stress 0caa elfiky in human shsy5y neuroblastoma in an autophagydependent manner peptidesdifferent peptides are used to target the cellsurface grp78 specifically peptides as anticancer drugs have two main types ishort naked peptides to induce apoptosis ii conjugated peptides todeliver an anticancer drug into cells for the first use grp78 serves as areceptor for the peptide and facilitates the internalization of the peptide which can then modulate various pathways peptides to induce apoptosisa gmbp1 peptidemultidrug resistance mdr is drug resistance that happens whencancer cells treated with one anticancer drug develop resistance todifferent drugs that are different from the used drug in structure andfunction an example of binding peptides that use grp78 as areceptor is gmbp1 which is used in reversing gastric cancer mdrgrp78 facilitates gmbp1 internalization into cells through the transferrinrelated pathway b gonadotropinreleasing hormone analogs gnrhagonadotropinreleasing hormone gnrha is a hypothalamus secreted hormone that affects sex hormones testosterone and estrogenmodified gnrha is more efficient than the natural form hence it is usedas a drug depending on the analog gnrha used as a drug againstendometriosis a case in which cells like that lining the inside of theuterus grow outside it in other parts of the body gnrha inhibitsproliferation and induces apoptosis of defected cells by inhibitinggrp78 thus leading to apoptosis conjugated peptidescell targeting is the solution for the nonspecific toxicity of anticancer drugs that affect cancer and healthy cells altogether and thusresulting in severe side effects peptides can target cancer cells anddeliver anticancer drugs into the cell in cancer cells the peptide canbind to the surface a membranebound form of the overexpressedchaperone grp78 to choose the peptide for a particular cancercell in vitro trials are required such as phage display phage display is atechnique for studying molecular interactions such as proteindnaproteinprotein and proteinpeptide utilizing the bacteriophages toencode peptides to genetic information a pool of cyclic peptidestested against the cancer cells and then a peptide is chosen to be usedfor drug delivery a pep42pep42 is a cyclic peptide ctvalpggyvrvc identified by thephage display technique against human melanoma cell line me66524 csgrp78 is the receptor for pep42 and facilitates it's internalization to the cell pep42taxol and pep42doxorubicin conjugates bind to grp78 in highly metastatic human melanoma cellsleading to its death in vitro leading to cancer cell death pep42selectively bind to grp78 and enter the cell and thus make it a powerfultool to deliver anticancer drugs to various cancer cells pep42 was used as a profiler for in silico predicting the csgrp78 andviral proteins of the zika virus human papillomavirus sarscov2and ebola virus b wifpwiql peptidewifpwiql peptide binds to grp78 expressed in breast cancer cellslife sciences surface in the breast and metastatic cells subtilase cytotoxin is a toxinfrom the ab5 toxins family subtilase cytotoxin composed of two subunits suba which is responsible for the toxicity and subb which isresponsible for subtilase cytotoxin internalization to the cell subatoxic effect is that it induces cell apoptosis by cleaving grp78 betweenthe amino acid residues leu416 and leu417 as indicated wifpwiql peptide binds to grp78 over cancer cells csgrp78 wifpwiqlsuba fusion resulting in an efficient anticancer agent wifpwiqlsuba works simultaneously wifpwiql is responsible for grp78recognition and internalization to the cancer cells while suba is responsible for the toxic effect on the cell by cleaving grp78 inside thecell and thus leading to apoptosis wifpwiql liposomes loadedwith doxorubicin are used to target csgrp78 overexpressed overvascular endothelial growth factor vegfactivated human umbilicalvein endothelial cells wifpwiql bound n2hydroxypropylmethacrylamide hpma copolymer aminohexylgeldanamycin conjugates were able to target csgrp78 and hence inhibit human prostatecancer cells additionally the genetic engineered mung beantrypsin inhibitor gbpti that includes the wifpwiql peptide wasable to induce apoptosis in colorectal cancer cells c bone metastasis targeting peptide bmtp78bmtp78 composed of a peptide wifpwiql conjugated withproapoptotic moiety dklaklak2 grp78 facilitates the internalization of bmtp78 into the cytoplasm in vitro trials showed thatbmtp78 induces apoptosis in human and mouse mammary cell lines dklaklak2 after internalization disrupts mitochondrial membranepermeability and thus kills the cell bmtp78 induced dosedependent cytotoxicity in human leukemia and lymphoma cell lines andacute myeloid leukemia patients additionally the grp78receptorbmtp78 system was used to image breast tumors accuratelythe adenoassociated virusm13derived phage aavp can be usedclinically to detect imaging and eradicate targeted therapy of inflammatory breast cancer utilizing csgrp78 as a target d girlrg peptidegirlrg is a peptide identified using phage display and bindsgrp78 girlrg conjugated to paclitaxelencapsulated nanopsspecifically targeted breast cancer and glioblastoma it was predicted in silico that girlrg binds to the atpase domain of grp78girlrg conjugated with poly ethylene glycol peg can efficientlytarget different tumor cell lines including heterotopic cervical ht3esophageal oe33 pancreatic bxpc3 lung a549 and glioma d54 additionally the radiolabeled 111inpeggirlrg show specificity toward cervical esophageal pancreatic lung and brain tumorsusing spect imaging e vap peptidesntrvap vap is a peptide identified using the phage displaytechnique and it binds to grp78 specifically sntrvap couplingwith a sirna for grp78 effectively downregulated its expression vap modified micelles rivap retro inverso isomer of lvapand dvap retro isomer of lvap could effectively achieve gliomatargeted drug delivery through grp78 at the same time it improvedthe therapeutic efficacy of paclitaxel for glioma binding peptides in diagnosticsas we mentioned before peptides could be used for drug delivery itcan be used as a carrier for radiolabels for imaging purposes such as inthe positron emission tomography pet utilizing the same concept oftargeting grp78 over cancer cellsradiolabeled polyethylene glycol peggirlrg is used in targetingmany cancers as heterotopic cervical esophageal pancreatic lung and 0caa elfiky glioma tumors triplenegative breast cancer tnbc resembles of breast cancer cases while the available diagnostic technologyfor its detection is by the invasive needle biopsy for example 68ga aradiolabel for pet imaging can be conjugated with dodecane tetraacetic acid dotavap grp78targeted pet imaging with [68ga]dotavap is a useful and accurate technique for imaging tnbc anddifferentiates it from other cancer types monoclonal antibodiesantibody ab also called immunoglobulin ig is a huge yshapedprotein produced mainly by plasma cells that are used by the immunesystem to neutralize pathogens such as viruses and bacteria the pathogenic molecule that is recognized by the antibody is called an antigen the antibody binds with the antigen with a keylock mechanism once the interaction established the cell bearing the antigentriggers a response such as metabolic inhibition monoclonal antibody mab159mab159 is a highly specific monoclonal antibody against the humangrp78 kd nm when administered mab159 found localized on the membranes of cancer cells but not normal celllinesupon glucose starvation stress mab159 is found more abundant on thecell membrane as the csgrp78 is pi3kakt signaling upstreamregulator through its interaction with crypto and alpha2macroglobulinover the cell membrane it is required for these factors to activate thepi3kakt signaling once bound to csgrp78 mab159 endocytosed and modulate the pi3k pathway leading to inhibition for cellproliferation tumor growth and metastasis at the same time it enhances tumor cell death both in vitro and in vivo the efficacy ofmab159 was examined in various tumor xenograft models includinght29 colon cancer h249 small cell lung carcinoma and a549 lungadenocarcinoma these cells have relatively higher surface grp78 expression compared to healthy cells mab159treatment led to and tumor growth inhibition in thesemodels respectively monoclonal igm antibody sam6the fully human monoclonal igm antibody sam6 was isolatedfrom a gastric cancer patient and it binds to an oglycosylated form ofgrp78 sam6 is internalized via endocytosis and is finally responsiblefor a lethal accumulation of oxidized lipoproteins followed by apoptosisin cancer cells sam6 not only bind to grp78 on the cancer cellmembrane but also it reduces the drug resistance and kills the cancercell human igm antibody patsm6patsm6 specifically binds to primary multiple myelomas cellsstaining the cells by immunohistochemistry reveals binding to grp78of the patsm6 this binding induces apoptosis and complementdependent cytotoxicity α 2macroglobulin α2mα2m is associated with the nterminal region of cellsurface grp78the binding activates akt to suppress apoptotic pathways and promotescell proliferation mouse mab c38 and c107the mouse monoclonal antibody c38 recognizes the cterminaldomain of the murine grp78 exposed on the cell membrane thebinding induces inhibition of the aktpi3k proliferative pathway inmelanoma cells a comparable experiment done on melanomamouse model shows that the antibody c107 also binds to grp78 inboth experiments the binging with the antibody decreases the tumrowth anti grp78cterminal domain ctd antibodies aretested against human prostate cancer cells it significantly reduceslife sciences tumor growth inhibits cell proliferation while promotes apoptosisbesides in the prostate cancer patients the antictd grp78 antibodybinds the cellexpressed grp78 in human prostate cancer cells conclusioncancerliketypes oftriplenegative breastgrp78 a master chaperone protein of the unfolded protein response plays an essential role in cancer chemoresistance and virulenceof the pathogenic infections targeting grp78 was utilized to defeataggressivecanceradditionally inhibiting grp78 overexpressed in viral infections issuggested as a promising strategy to reduce the virulence of manyviruses and fungal infections the present review summarizesthe up to date targeting strategies used to inhibit cellsurface grp78illuminating the potential use of these strategies to defeat both cancerchemoresistance and viral and fungal infectionsdeclaration of competing interestall the authors declare no conflict of interest for this workreferencesmol cell biol l ellgaard a helenius quality control in the endoplasmic reticulum nat rev mj gething j sambrook protein folding in the cell nature h hu m tian c ding s yu the cebp homologous protein chop transcription factor functions in endoplasmic reticulum stressinduced apoptosis andmicrobial infection front immunol im ibrahim dh abdelmalek aa elfiky grp78 a cells response to stress lifesci e little m ramakrishnan b roy g gazit as lee the glucoseregulatedproteins grp78 and grp94 functions gene regulation and applications critrev eukaryot gene expr l brocchieri ec de macario aj macario hsp70 genes in the human genomeconservation and differentiation patterns predict a wide array of overlapping andspecialized functions bmc evol biol am ismail aa elfiky wm elshemey recognition of the gluconeogenic enzyme pck1 via the gid4 e3 ligase an in silico perspective j mol recognit e2821 pc liao sk tan ch lieu hk jung involvement of endoplasmic reticulum inpaclitaxelinduced apoptosis j cell biochem i haas bipa heat shock protein involved in immunoglobulin chain assemblycurr top microbiol immunol j li m ni b lee e barron d hinton a lee the unfolded protein responseregulator grp78bip is required for endoplasmic reticulum integrity and stressinduced autophagy in mammalian cells cell death differ s srivastava g jain s dang s gupta r gabrani phytochemicals targetingendoplasmic reticulum stress to inhibit cancer cell proliferation anticancerplants natural products and biotechnological implements springer pp r ge c kao cell surface grp78 as a death receptor and an anticancer drugtarget cancers u gopal sv pizzo the endoplasmic reticulum chaperone grp78 also functionsas a cell surface signaling receptor cell surface grp78 a 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modulatesautophagosome biogenesis nat commun x yuan m dong x li j zhou grp78 promotes the invasion of pancreatic aa alhashimi j rak rc austin cell surface grp78 a novel regulator of 0caa elfiky life sciences f visioli y wang gn alam y ning pv rados je nör glucose a choukhi s ung c wychowski j dubuisson involvement of endoplasmic yl tsai as lee cell surface grp78 anchoring and translocation mechanisms ch ji hy kim aj heo sh lee mj lee sb kim the ndegron p mehrbod sr ande j alizadeh s rahimizadeh a shariati h malek pathway mediates erphagy mol cell 1072e9the roles of apoptosis autophagy and unfolded protein response in arbovirusinfluenza virus and hiv infections virulence and therapeutic potential in cancer cell surface grp78 a new paradigm insignal transduction biology elsevier pp yj chang yp huang zl li ch chen grp78 knockdown enhancesapoptosis via the downregulation of oxidative stress and akt pathway afterepirubicin treatment in colon cancer dld1 cells plos one e35123 t chen s xu chronic exposure of cisplatin induces grp78 expression in ovariancancer proceedings of the 4th international conference on biomedical andbioinformatics engineering acm y kim am lillo sc steiniger y liu c ballatore a anichini targetingheat shock proteins on cancer cells selection characterization and cellpenetrating properties of a peptidic grp78 ligand biochemistry z li l zhang y zhao h li h xiao r fu cellsurface grp78 facilitatescolorectal cancer cell migration and invasion int j biochem cell biol z niu m wang l zhou l yao q liao y zhao elevated grp78 expression isassociated with poor prognosis in patients with pancreatic cancer sci rep s tian w chang h du j bai z sun q zhang the interplay betweengrp78 expression and akt activation in human colon cancer cells under celecoxibtreatment anticancer drugs j xie zh tao j zhao t li zh wu jf zhang glucose regulatedprotein grp78 inhibits apoptosis and attentinutes chemosensitivity of gemcitabine in breast cancer cell via aktmitochondrial 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since leung reported a single case of lowdose aspirin lda induced multiple smallintestinal ulcers in many investigators have described ldainducedsmall intestinal mucosal injuries watanabe reportedthat all of their patients who used lda had small intestinallesions that were detected using capsule endoscopy 0cgastroenterology research and practicece iwamoto investigated patients whounderwent ce for occult bleeding and erosions wereobserved in cases of whom were taking lda ornonlda nonsteroidal antiammatory drugs endo described small intestinal lesions in of subjects who took aspirin mg for weeks shiotani performed ce on young healthy individuals beforeand after medium doses of enteric aspirin were administered for days and rabeprazole mg was administered for week and found large erosions that included small intestinal ulcers in of the subjects recently direct oral anticoagulants doacs have beenadministered as alternatives to warfarin dabigatran is adirect thrombin inhibitor and rivaroxaban apixaban andedoxaban are factor xa inhibitors doacs have significantlyfewer side eï¬ects than warfarin including intracranial hemorrhage hence the number of patients taking doacs isgradually increasing [] howeverthe ï¬ndings frommetaanalyses have shown that compared with warfarinthe incidence of gastrointestinal gi bleeding is higher inassociation with doacs [ ] the causes of gi bleedingin association with doacs were bleeding from colon astric cancers and diverticular hemorrhages comparedwith warfarin dabigatran and rivaroxaban are associatedwith higher risks of gi bleeding depending on their dosesand the risk of gi bleeding associated with apixaban iscomparable edoxaban is associated with significantlyless gi bleeding at low doses mg once daily comparedwith warfarin but the risk is significantly greater at highdoses mg once daily but unlike western countries the rate of gi bleeding for both dabigatran and rivaroxaban is equivalent to warfarin in asian countries furthermore edoxaban tends to cause less digestive tractbleeding than warfarin esophageal ulcers caused bydabigatran have been described by toya kasai and okada and okada the tartaric acidcoating on dabigatran causes esophageal mucosal disorders because dabigatran persists in the midesophagus ifit is consumed without water however there havebeen no reports of smalllesions in patientswho receive doacs here we aimed to evaluate smallintestinal mucosal injuries in patients taking doacs usingvideo capsule endoscopy vceintestinal methodsthis study was a prospective openlabel nonblinded multicenter and observational study from september tomarch pat5ents taking doacs namely dabigatranrivaroxaban and apixaban for atrial ï¬brillation at saitamamedical university hospital keio university hospital saitama medical center and yokohama municipal citizenshospital were enrolled patients with severe comorbiditiesincluding severe anemia and exacerbations of heart failurethat required blood transfusion crohns disease and ileuswere excludedthe hemoglobin hb and serum ferritin levels the esophagogastroduodenoscopy egd ï¬ndings and colonoscopicï¬ndings were examined vce pillcam sb2 given imagingfigure rednessfigure erosionltd yoqneam israel was performed to examine small intestinal lesions according to the doac used redness erosionulcer and angioectasia were checked redness was a red spotfigure and erosions were deï¬ned as small and superï¬cialmucosal disruptions denuded of villi figure ulcers weredeï¬ned as large submucosal disruptions with a central areacovered with exudate and angioectasia was a patchy erythematous lesion figure the images were analyzed using theproprietary rapid software by an expert n h whohad performed more than vce examinations blindlythe type and location of smallbowel lesions were registeredalso the proportion of lesions detected between types ofdoac was evaluated and the hemoglobin hb and serumferritin levels were compared between patients with and without smallbowel lesionsthe study protocol accorded with the tenets of therevised declaration of helsinkiand it wasapproved by the institutional review boards at our institutions written informed consent was obtained from all ofthe patients this study was registered with the universityhospital medicalinformation network clinical trialsregistry umin000011527 october 0cgastroenterology research and practicetable patients characteristicsparametermean age years rangesex n malefemalecomorbid disease natrial ï¬brillationparoxysmal atrial ï¬brillationhypertensionhyperlipidemiacerebral infarctiondoac n years dabigatranrivaroxabanapixabanbayaspirin ncelecoxib nppi nh2 blocker n mean hb gdl rangedoac direct oral anticoagulant ppi proton pump inhibitor hbhemoglobinlower portion in patients table erosions wereobserved in patients and they were present in theupper portion in in the middle portion in and in the lower portion in patientstable erosions tended to occur less frequently in themiddle portion however the diï¬erence was not significantp compared with the upper portion p compared with the lower portion angioectasia was observedin patients and was present in the upper portionin patients and in the middle portion in patient and was absent from the lower portion table there were no ulcers in any patients erosions tended to bemore frequent in patients taking dabigatran or apixaban thanin patients taking rivaroxaban this diï¬erence was not significant p table no significant diï¬erences wereobserved regarding angioectasia among the patients takingthe diï¬erent doacs none of these patients had activebleeding from small intestinal lesionsthe mean hb concentrations in the patients with andlesions were gdl and gdlwithout smallbowelrespectively a diï¬erence which was not significant p the mean ferritin levels in the patients with and withoutsmallbowel lesions were mgdl and mgdl respectively a diï¬erence which was not significant p discussionthis studys ï¬ndings showed that of the patients who tookdoacs had redness had erosionsor small ulcers had angioectasia and had no abnormalities in their small bowel smallbowellesions were observed in of patients thereforethere was a high incidence of smallbowel lesions in patientsfigure angioectasiaibm®spss® statistical software version ibm corporation armonk ny usa was used for the statisticalanalyses the data were analyzed using ttests and fishersexact test resultsthirtythree patients were enrolled to participate in thisstudy but patients withdrew their consent and vce wasperformed on patients the patients mean age was years range years and males and females participated in this study twenty patients had atrial ï¬brillation had paroxysmal atrial ï¬brillation had hypertension hadhyperlipidemia and had cerebral infarction eight patientstook dabigatran took rivaroxaban and took apixabanthe mean duration of doac use was months months additionally patient took bayaspirin patienttook celecoxib patients took proton pump inhibitorsppis and patients took h2 receptor antagonists theaverage hb concentration was gdl range gdl table twentytwo patients underwent egdand atrophic gastritis was present in patients hiatal hernias in patients gastric polyps in patients erosive gastritisin patients gastric ulcer or ulcer scar in patients reï¬uxesophagitis in patients endoscopic submucosal dissectionscar for early gastric cancer in patients and an esophagealulcer in patient nineteen patients underwent colonoscopyand colonic polyps were present in patients colonic diverticulum were present in patients and a rectal ulcer waspresent in patient none of these lesions detected by egdand colonoscopy had active bleedingthe patients evaluated with vce was redness in thelower esophagus was present in patient gastric erosionswere present in patients and gastric redness was presentin patient table the patient who had redness in thelower esophagus was taking apixaban smallbowel transitwas complete in of patients smallbowellesions were observed in of patients redness was observed throughout the small intestine in patients and it was present in the upper portionin in the middle portion in and in the 0cgastroenterology research and practicetable capsule endoscopy ï¬ndingsentire small intestine observation rate n detection rate n esophagusstomachupper small intestinemiddle small intestinelower small intestineentire small intestineredness lower n erosions n redness n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions nangioectasia nno abnormalities n table findings at each small intestinal site according to the directoral anticoagulant useddabigatranrivaroxabanapixabansiteï¬ndingupper small intestineredness nerosions nangioectasia nmiddle small intestineredness nerosions nangioectasialower small intestineredness nerosions nangioectasia nentire small intestineredness nerosions nangioectasia ntaking doacs however none of these patients had activebleeding and most of the lesions were mild patients withsevere anemia or active bleeding were excluded from thisstudy hence only patients with mild symptoms wereincluded ldainduced lesions cause redness erosions andulcers [] previous studies ï¬ndings that describe the characteristics of smallbowel injuries associated with chroniclda use suggest that ulcers are observed mainly in the distalpart of the small bowel [] in this study erosionstended to be observed less frequently in the middle portionof the small bowel in the patients taking doacs howeverthere were no significant diï¬erences regarding the distributions of the lesions there were no ulcers in any patientstherefore the intake of doac might not be related withsevere ulcers in the small intestinein this study angioectasia was observed in the upper andmiddle portions but not in the lower portion of the smallbowel kaufman used a transit timebased quartilemethod to evaluate patients with angioectasia whounderwent ce and found that most lesions were inthe ï¬rst quartile igawa reported that while therewere no diï¬erences regarding the location of type 1a angioectasia among patients with occult gastrointestinal bleedingtype 1b angioectasia was relatively less frequent in the lowerportion compared with that in the upper and middle portionsof the small bowel the data reported before thereforeangioectasia might not be aï¬ected by the intake of doacscomparison of vce ï¬ndings before and after the administration of d719acs is neededno significant relationships were determined in relationto the presence of the hb level or the serum ferritin levelbetween the patients with and without smallbowel lesionsin this study despite detecting abnormal ï¬ndings in thesmall bowel no active bleeding was seen by vce and therewas no severe anemia in any patients in this study furthermore compared with warfarin the incidence of gi bleedingis higher in association with doacs the causes of gi bleeding in association with doacs are bleeding from colon astric cancers and diverticular hemorrhages however noneof these lesions detected by egd and colonoscopy had activebleeding doacs did not aï¬ect bleeding from the upper gitract and the colon in this studyno significant diï¬erences were observed among thedoacs in relation to smallbowel lesions the ï¬ndings fromthe randomized evaluation of longterm anticoagulationtherapy trial of dabigatran showed that in the warfaringroup patients with gi tract bleeding had gastric canceror colonic cancer and that in the dabigatran group patients with gi tract bleeding had colonic cancer and patient had gastric cancer hence dabigatran mightinduce gi tract bleeding from colon cancer rivaroxaban apixaban and edoxaban compete directly with the s1pocket of factor xa and inhibit factor xa activity whereasdabigatran is a prodrug that is activated in the presence ofesterase in the gi tract plasma and liver the causes of themucosal damage by dabigatran were thought to be due todirect acting at the local area where it is absorbed in addition tartaric acid coats dabigatran tablets and the tabletscan cause mucosal damage if they are retained within theesophagus while we expected an increase in the frequencyof intestinal mucosal injury among the patients who tookdabigatran as a consequence of the tartaric acid coating this 0cgastroenterology research and practicestudys ï¬ndings did not demonstrate a higher rate of smallbowel lesions associated with the use of this doac themechanisms underlying mucosal injuries caused by doacsother than dabigatran remain unclear smallbowel lesionsincluding redness erosions and angioectasia might be moreeasily detected by performing ce on patients who takedoacs because the doacs might cause bleeding thatcould facilitate the detection of the lesionsthis study has several limitations while this was a multicenter study the sample size was small hence more patientsshould be accrued and investigated in the near future moreover this study only included data that described the patientsï¬ndings after the administration of the doacs and datadescribing the ï¬ndings before their administration wereabsent therefore it remains unclear whether small intestinallesions are directly induced by doacs studies of patientsï¬ndings before and after the administration of doacs areneeded in the near future furthermore patients with severeanemia and overt bleeding were excluded from this studyand most of the enrolled patients did not have bleeding orhad minor bleeding patients taking edoxaban were notincluded in this study because edoxaban was not available injapan when this study began conclusionlesionssmallbowelincluding redness erosions smallulcers and angioectasia were detected in of patientswho took doacs more patients using doacs should beinvestigated using ce in the near futureabbreviationsdoac direct oral anticoagulantvideo capsule endoscopyvceldalowdose aspirincapsule endoscopycegastrointestinalgihemoglobinhbegdesophagogastroduodenoscopyproton pump inhibitorppidata availabilitythis manuscript describes a study that was aimed at evaluating direct oral anticoagulant doac related to smallbowellesions using video capsule endoscopy we believe that ourstudy makes a significant contribution to the literaturebecause its ï¬ndings showed that many patients takingdoacs had smallbowel lesions however most lesions wererelatively mild and they did not cause bleedingconflicts of interestthe authors have no conï¬icts of interest to disclose that arerelevant to this studyauthors contributionshiroyuki imaeda was responsible for the conception anddesign and ï¬nal approval of the article minoru yamaokahiroyuki imaeda naoki hosoe kazuaki yoneno ryukanno hisashi mitsufuji takahiro sasaki and keijiyamamoto were responsible for enrollment of patientshiroyuki imaeda and naoki hosoe were responsible foranalysis and interpretation of the data minoru yamaokaand hiroyuki imaeda were responsible for drafting of thearticle naoki hosoe was responsible for the critical revision of the article for important intellectual content takanori kanai toshimasa yamamoto toshihide mimuraharuhiko ogata nobuo araki and hidetomo nakamotowere the supervisorsreferences w k leung i bjarnason v w s wong j j y sung andf k l chan small bowel enteropathy associated withchronic lowdose aspirin therapy lancet vol no t watanabe s sugimori n kameda small bowelinjury by lowdose entericcoated aspirin and treatment withmisoprostol a pilot study clinical gastroenterology andhepatology vol no pp j iwamoto y mizokami y saito smallbowel mucosalinjuries in lowdose aspirin users with obscure gastrointestinalbleeding world journal of gastroenterology vol no pp h endo k hosono m inamori incidence of smallbowel injury induced by lowdose aspirin a crossover studyusing capsule endoscopy in healthy volunteers digestionvol no pp a shiotani k haruma r nishi randomized doubleblind pilot study of geranylgeranylacetone versus placebo inpatients taking lowdose entericcoated aspirin lowdoseaspirininduced small bowel damage scandinavian journalof gastroenterology vol no pp s j connolly m d ezekowitz s yusuf dabigatranversus warfarin in patients with atrial ï¬brillation the newengland journal of medicine vol no pp m r patel k w mahaï¬ey j garg rivaroxaban versuswarfarin in nonvalvular atrial ï¬brillation new england journal of medicine vol pp c b granger j h alexander j j v mcmurray apixaban versus warfarin in patients with atrial ï¬brillation thenew england journal of medicine vol no pp c t ruï¬ r p giugliano e braunwald comparison ofthe eï¬cacy and safety of new oral anticoagulants with warfarinin patients with atrial ï¬brillation a metaanalysis of randomised trials lancet vol no pp c s miller a dorreen m martel t huynh and a n barkun risk of gastrointestinal bleeding in patients taking nonvitamin k antagonist oral anticoagulants a systematic reviewand metaanalysis clinical gastroenterology and hepatologyvol no pp 1683e3 d caldeira m barra a ferreira systematic reviewwith metaanalysis the risk of major gastrointestinal bleeding 0cgastroenterology research and practicewith nonvitamin k antagonist oral anticoagulants alimentary pharmacology therapeutics vol no pp t yamashita y koretsune y yang edoxaban vs warfarin in east asian patients with atrial ï¬brillationan engageaftimi subanalysis circulation journal vol no pp m hori m matsumoto n tanahashi rivaroxaban vswarfarin in japanese patients with atrial ï¬brillation circulation journal vol no pp y toya s nakamura k tomita dabigatraninducedesophagitis the prevalence and endoscopic characteristicsjournal of gastroenterology and hepatology vol no pp k kasai e ishida and y kobayashi two cases of esophagealulcer caused by dabigatran gastroenterological endoscopyvol pp m okada and k okada exfoliative esophagitis and esophageal ulcer induced by dabigatran endoscopy vol supplement pp e23e24 t vanassche j hirsh j ginsberg and j eikelboom anspeciï¬c bleeding patterns of anticoagulant therapy lessonsfrom clinical trials thrombosis and haemostasis vol pp h endo k hosono m inamori characteristics ofsmall bowel injury in symptomatic chronic lowdose aspirinusers the experience of two medical centers in capsule endoscopy journal of gastroenterology vol no pp i watari s oka s tanaka comparison of smallbowelmucosalinjury between lowdose aspirin and nonaspirinnonsteroidal antiammatory drugs a capsule endoscopystudy digestion vol no pp h endo k hosono t higurashi quantitative analysisof lowdose aspirinassociated small bowel injury using a capsule endoscopy scoring index digestive endoscopy vol no pp d kaufman g leslie n marya smallintestinalangioectasia characterization risk factors and rebleedingjournal of clinical gastroenterology vol no pp a igawa s oka s tanaka major predictors and management of smallbowel angioectasia bmc gastroenterologyvol no 0c' | 0 |
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14 cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classiï¬ed into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in speciï¬c chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22 ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and ï¬broblastgrowth factor receptor FGFR gene fusions30 Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients beneï¬t from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classiï¬cation andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aï¬atoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver ï¬ukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difï¬culty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespeciï¬c survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Paciï¬c Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11 Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14 Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is aï¬rm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was ï¬rst described in cHCCICC was not systematicallydescribed until when it was classiï¬ed into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly deï¬ned areas type C mixedtype presents as intimate association without clear boundaries18 In another classiï¬cation system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identiï¬able intermediatetransition between HCC and ICC type III ï¬brolamellar tumors resemblesthe ï¬brolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classiï¬cationis commonly used in which cHCCICC is classiï¬ed into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a uniï¬ed classiï¬cation system greatly adds to the difï¬culty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdeï¬ned cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identiï¬ed stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA ampliï¬cations are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in of cases Recently some oncogenic genes were identiï¬ed in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identiï¬ed as a driver ofHCC45 VEGFA and CCND1FGF19 have also identiï¬ed in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identiï¬ed For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22 and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately of cases22 Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30 Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in of ICC cases32 Fusions ampliï¬cations translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC and ICC Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate ï¬lament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difï¬cult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166 Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efï¬cacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L ï¬rstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5ï¬uorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71 This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current ï¬rstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onï¬rstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand ï¬uoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the ï¬rstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe beneï¬t of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] conï¬dence intervalPFS months versus months p This effectiveness wasconï¬rmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the ï¬rstlinetreatment for advanced cholangiocarcinoma[CI] and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe ï¬rstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efï¬cacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efï¬cacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased beneï¬t for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095 Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI months CI months CI and months CI respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as ï¬rst line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir ï¬rstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the ï¬rst US Food and Drug Administration FDAapproved ï¬rstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPaciï¬c have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the ï¬rst generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have beneï¬tedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1 FGFR1 PDGFR RET and KIT In August theFDA approved lenvatinib for ï¬rstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the ï¬rstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modiï¬cations Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can speciï¬cally inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPaciï¬cREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further conï¬rmed the efï¬cacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival beneï¬t observed across all age subgroups and atolerable safety proï¬le supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety proï¬le apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic proï¬le and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a ï¬rstinhuman phase I study NCT02508467 of ï¬sogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123 Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identiï¬ed in recent years is the inhibitor of the ï¬broblast growth factorFGF signaling pathway which consists of members labeled FGF1FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1 FGF signals regulate cell proliferation in which FGFR2fusions occurred in of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the ï¬rst and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on ï¬ndings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may beneï¬t from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPï¬zeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial Identiï¬erTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi | 2 |
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