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this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy — group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy — the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of — cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples ˆ’ a blanka control ˆ’ a blank — the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisˆ’b ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa —\uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of — cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of — viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy — fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at ˆ’°c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aˆ’ba — where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight — histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlich™s hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field — molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturer™s instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ˆ†ˆ†ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ˆ’ˆ†ˆ†ct ˆ†ˆ†ct is the difference between the mean ˆ†ctsample and mean ˆ†ctcontrol where ˆ†ctsample is the difference between the mean ctsample and the mean ctβactin and ˆ†ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferroni™s posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a — also it displays intact cancer cells arrow and giant cells arrow figure 3b and c — the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d — also displays a notable necrosis of cancer cells n figure 3e — the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ” ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m — and — a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f — and 4g — however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h — and 3i —the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy — andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101aˆ’ ± 217abˆ’ ± 201bˆ’ ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1
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" Despite several efforts the development of an effective vaccine for COVID19 may take a much longer timeTraditionalnatural medicine already experienced by humans could be an earlier solution Considering the researchteam™s experience in using nanoclays as highaffinity material for cancer metastasis melanoma treatment andbone regeneration we propose to use these nanoclays for the preventiontreatment of COVID19 Owing to highaffinity nanoclays would capture the viruses before the latter get engaged with human hACE2 In this studymolecularlevel simulations and modeling of the interaction of coronavirus spike and hACE2 proteins wereperformed with and without nanoclays The results showed a very high level of affinitycohesiveness among SARSCoV2 spike and nanoclays as compared to the one between the former and hACE2 We premise that these nanoclays since already being used as drug carriers could also be injected as œclaysalone medicine Recommendationshave also been provided for future in vitro and in vivo studiesBackgroundThe sudden emergence and rapid spread of novel coronavirus SARSCoV2 have significantly affected thehealth and lives of human beings in addition to criticallyaffecting the world economy SARSCoV2 spike S bindswith high affinity to human angiotensinconverting enzyme hACE2 and uses it as an entry receptor to invade target cells Fig 1a b [] The virussurface spikeprotein mediates coronavirus entry into host cellsSARSCoV2 spike protein contains a receptorbindingdomain RBD that recognizes explicitly as its receptorhACE2 [ ] The surface of hACE2 contains two virusbinding hotspots that are criticalfor SARSCoV2 Sbinding Several naturally selected mutations in SARSCoV2 RBD surround these hotspots and regulate theinfectivity pathogenesis and crossspecies and humantohuman transmissions of SARSCoV2 [ ]At present there are no clinically approved vaccinesor drugs that specifically target SARSCoV2 Followingthe real protocol of developing a vaccine it may takemuch longer time to come up with an effective vaccine Correspondence habibrehmankfupmedusa3Engineering Research International ERI Riyadh Saudi ArabiaFull list of author information is available at the end of the There is a lot of interest in the development of therapeutic antibodies against SARSCoV2 Despite many efthese antibodies have not yet beenforts howeverdiscovered [] exceptin a few trials [] One trialshowed the potent neutralization of SARSCoV2 bybinding to the RBD of its S glycoprotein [] In this trial[] antibody cocktails a mixture of different antibodiesis recommended due to the increased neutralization effect it has on SARSCoV2 However use of antibodiesin the past from convalescent patients of SARSCoV totreat SARSCoV infection has shown adverse reactionsin the patients such as AntibodyDependent Enhancement ADE causing increased viral infectivity and otherharmful immune responses [] Moreover based on theexperience with the vaccine development efforts forSARSCoV and MERS chances of the materialization ofthe efforts being made for SARSCoV2 seems quitethin Therefore naturaltraditional medicines that have ahistory of safe consumptioningestion by humans couldbe considered as one of the treatment options for SARSCoV2 Being a natural material and a history of humanuseconsumption we suggest œhighly charged nanoclays to be used as coronavirus blockers and inhibitorsof the spikemediated entry into the human cells 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 httpcreativecommonslicensesby40 0cAbduljauwad Nanoscale Research Letters Page of Fig Schematics of the SARSCoV2 attack on human hACE2 and the subsequent immune system response a b RBD binding hACE2 withoutan interference c RBD complexed with the antibody at receptor attachment site hence competing with hACE2 d RBD complexed with RBD at asite other than where receptor attaches resulting in the alteration of RBD structure and interruption of lock and key binding of RBD to hACE2Nanoclays nanosized natural materials originatingfrom minerals of the sedimentary rocks have got avery high affinity to bacteria and viruses [] Due toisomorphous substitution in their molecular structurethese nanoclays exhibit charge deficiency on theirsurfaces This charge deficiency on their surfaces isneutralized by the water molecules and the dissolvedcations Fig The charged structure and large surface area of clay nanops give them an affinityfor charged entities as found on bacterial surfacesand bacterial toxins Their distinct biomedical properties include high absorption the ability to engulf microbes and no toxicity Each of the electrically activeclay minerals has its distinct morphology characteristics and interaction behavior The most studied biomedical application of nanoclays includes serving ascarriers and complexes for anticancer drugs such as5fluorouracil and trastuzumab [“] They havetherefore been a potential alternate medicine for several diseases [“] Clay nanops due to theiradhesive nature have also been used as carriers forsustainedrelease medicine [ ] Nanoclays havealso successfully been used to adsorb and treat bovinerotavirus and bovine coronavirus [] Researchers[] intercalated methotrexate MTX an anticanceragentinto the anionic clay to create a nanohybriddrug They used the coprecipitation and subsequenthydrothermal methodology to prepare this chemicallystructurally and morphologically welldefined twodimensional drugclay nanohybrid The researchers[] discovered that due to the biocompatibility andhigh loading capacity bentonite nanoclay could beused for the preparation of the drugdelivery vehiclesthey prepared doxorubicinbentoniteIn thisto form ananoclay complex DOXBent complexsustainedreleaseintradrugdeliverystudysystem for 0cAbduljauwad Nanoscale Research Letters Page of Fig a SEM image and b the corresponding molecular structure of Namontmorillonite showing the configuration isomorphous substitutioncharge deficiency and interlayer cations from []tumoral chemotherapy of melanoma As montmorillonite clay is recently being studied to be used as anadditive and drug carrier materialthese nanoclaycomposites appeal their use in various dosing formmainly for controlled release of the drug [] The researchers [] also discovered that nanoclays can beused into recent dual functional drug delivery systemsDDSs to have efficiency in the drug delivery and soreduce the toxicity of doxorubicin DOX that is being used for thyroid cancer treatment Using a libraryof single“single type photo cleavable amphiphilicJanus dendrimers researchers [] developed a selfassembling lightresponsive dendrimersomes vesicleplatform Similar to the nanoclays surface modifiedbioactive virusmimicking anic nanovesicles fromglycodendrimersomes have structural modificationsthat contribute to manifest SARSCoV2 and hostpathogenic molecular interactions that help the virusto escape from the human immune system []Through considerable previous research we developedbasic characterization and behavior modeling ofthecharged clay minerals [“] and their applications in thecontrol of cancer metastasis [] in vitro and in vivo studies on melanoma treatment [] and the calcium depositionbone regeneration studies [] In a previous study bythe authors [] it was demonstrated that clay nanops had got a high affinity to the charged surfaces Thehigh attraction affinity of the nanoclays and the increasednonspecific adhesion attraction of the cancer cells makenanoclays favorable candidates to control cancer metastasis In that study we demonstrated the possible use of twocharged clay minerals to control the metastasis of thecancer cells Namontmorillonite SWy3 and palygorskitePFll Further to the findings of the authors™ previous research [] on the use of these nanoclays for the control ofcancer metastasis we also through in vitro and in vivostudies established that these nanoclays have inhibitory effects on melanoma cancer cells mainly on cell proliferationand viability [] In these previous studies in addition tolaboratory experiments molecularlevel simulations werealso performed on the nanoclay and cells™ interactionsThese simulations provided the assessment of the relativelevel of cohesivenessaffinity in the interactions with andwithout clay nanopsperceptionthroughthisestablishingBased on all the above experience of the authors onthe highaffinity potential of nanoclays we propose thatthe nanoclays could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2 This paper is a first steptowardsamolecularlevelsimulation and modeling approachBased on the results of the molecularlevel simulationsan outline of the recommendations for the next phasesof in vitro and in vivo research is also provided As thesenanoclays are also successfully being used as medicinecarriers we also premise that they can also be injectedingested as œclaysalone medicine and thus we haveproposed a tentative nanoclays administration methodology for this purposeMaterials”MoleculesSelection and Formulation of SARSCoV2 and hACE2Molecules of SARSCoV2 spike S and hACE2 were acquired from the protein data bank website RCSB [“] 0cAbduljauwad Nanoscale Research Letters Page of The molecular models of SARSCoV2 spike S andhACE2 formulated in Materials Studio software [] arerespectively shown in Fig 3a b Before being subject tothe simulations these molecules were charged using thecharge equilibration method QEq of the softwareSelection and Formulation of Nanoclay CrystalliteNamontmorillonite one of the most active members ofthe smectite group of clay minerals was selected for thestudy Namontmorillonite is a layered phyllosilicate claysmectite Fig In the colloidal form the space between neighboring layers can contain free sodium calcium or magnesium cations that are electrostaticallyattracted to external negatively charged surfaces [] Inits dry powdered state Namontmorillonite exists asequidimensionalflakessheets with dimensions of approximately × × microns Fig 2a Thesenegative charges on their interlayer surfaces are balancedby the cations As colloids the interlayer cations get dissociated from the clay ps and associate themselveswith the other negatively charged surfaces These ps also have positively charged edges due to the presence of the broken bonds at their ends Morphology andfurther characteristics of these nanoclays are providedin Table while formulation of their crystallites in Materials Studio software are explained belowIn the software Namontmorillonite crystallites wereformulated based on fundamental properties such asCEC exchangeable cations and interlayer charges Table The size of the molecularcrystallite size was selectedbased on the results of the p size analysis usingthe dynamic light scattering DLS technique [] Thefinal form of clay crystallite created in the software istheseshown in Fig 3c Afterthe preparation ofcrystallites in the design mode of the software using theinherent properties these were charged using the chargeequilibration method QEq of the softwareMethods”MolecularLevel SimulationsThis part of the study consisted of the simulation andassessment of the interactions of the SARSCoV2 spikeS with clay crystallites and with hACE2 Although thesemodels may not be the complete replication of the actual in vitro conditions these have been incorporatedwith all the essentialinteractions and are quite wellsuited for the intended relative and comparative studyIn the software the sorption and simulations of theformulated configurations of SARSCoV2 S Namontmorillonite crystallites and hACE2 were carriedout using Monte Carlo MC and molecular mechanicsMM techniques The enhancement of affinity in all thesimulated configurations was assessed in terms of thecalculated cohesive energy density CED”CED beingconsidered as a measurement of the cohesiveness of themolecular system Due to the largesized computationsinvolved in the simulationsthese calculations werecarried out using the highperformance computing facilities HPC at KFUPM KSA The overall methodologyand the choice of particular methods and the simulationparameters were based on authors™ previous research[“] while it is detailed in the subsequent sectionSARSCoV2 Spike S Interactions with hACE2 and ClayCrystallitesTo simulate the interaction of SARSCoV2 S with claycrystallites various numbers of the crystallites of Namontmorillonite clay were sorbed on SARSCoV2 Smodel For these sorption simulations the MetropolisFig Molecularlevel models of a SARSCoV2 spike b hACE2 and c Namontmorillonite crystallite formulated in Materials Studio software 0cAbduljauwad Nanoscale Research Letters Page of lnoitauccoFlnoitcaretnInoitcaretnInoitcaretnIninoisrepsidretawnoisrepsiDygrenelatotygreneWdvˆ’ygreneBAlraopcilihpordyHˆ’ˆ’ˆ’ˆ’aCaNretaWytiniffasγlaitnetopVmPZateZreyalretnIegrahclardeharteTlardehatcOlebaegnahcxEegrahcegrahcsnoitacqemgCECecafruSNaeragmslarenmirehtOliacmehClaumrofacilisOiSgMlAaCaNecruoSytnuoCASUYWkoorC][yWSyacletinolliromtnomaNfonoitaziretcarahcliacmehcdnalacisyhpfoyrammuSelbaT 0cAbduljauwad Nanoscale Research Letters Page of Monte Carlo method was selected in the Sorption module of the software In each sorption step clay crystallitesoccupy spaces around the spike S model to lower theoverall energy of the complex The required number ofcrystallites were sorbed in a maximum of stepsand then the energy of the system was minimized usingthe Forcite module of the software based on the MDprinciples The similar sorption process was repeated forthe interaction modeling of the SARSCoV2 spike molecule with hACE2 In this process hACE2 moleculeswere sorbed around the RBD of the spike S of SARSCoV2 After the completion of the sorption process theenergy of the formulation was minimized using MDbased module of the softwareThe Forcite module ofthe software incorporatingNPT constant number of ps pressure andtemperature ensemble was used for MD simulationswith a modified universal force field [] The simulations were run for to ps with an interval of 05fs ortill a constant volume is obtained A Berendsen thermostat with a decay constant of ps was used to controlthe temperature during the simulation During the MDsimulations the assumed temperature was kept constantat K °C with an atmospheric pressure kPaA Berendsen barostat with a decay constant of pswas used to control the pressure of the system TheBerendsen methodology was considered as the most appropriate for the single crystallites after several trials involving other thermostats and barostats available in thesoftware In the Monte Carlo method the parametersfor the ratios of exchange conformer rotate translateand regrow were selected as and respectively with the corresponding probabilities as and Amplitudes adapted for rotationand translation were ° and respectivelyCohesive Energy Density CED MeasurementIn this study the assessment of the affinitybindinglevelin the SARSCoV2clay crystallites and SARSCoV2hACE2 complexes was measured through thechanges in the CED After the sorption of clay crystallites and the subsequent performance of moleculardynamics of each of the configurations the CED wasdetermined using the cohesive energy density optionof the Forcite module of the software The authorshave experienced that the CED concept consisting ofthe total van der Waals and electrostatic CEDs canquite closely explain the various molecularlevel processes and interactions and simulate the extent of affinitybinding created among the simulated complexes[“] Quantitatively CED is defined as the amountof energy needed for the transition of mol of material from the liquid to the gaseous phase It is also ameasureofaffinityattractivenessthe mutualofmolecules and is expressed both as electrostatic andvan der Waalsan NPTensembleaveraged overforcesIn the Forcite module van der Waals energies wereevaluated using atombased cutoffsIn this methodnonbond interactions are simply calculated to a cutoffdistance and interactions beyond this distance are ignored To avoid the discontinuities caused by direct cutoffs most simulations use a switching function to turnoff nonbond interactions over a range of distancessmoothly An effective potential is created by multiplyingthe actual potential by the smoothing function Thechoice of the function in the intermediate range is crucial and should be continuously differentiable in this region so that forces can be calculated In this study acubic spline smoothing function was used with a splinewidth of and a cutoff distance of Results and DiscussionsThe final configuration of the SARSCoV2 ShACE2complex is shown in Fig 4a while the complexes between SARSCoV2 spike and different numbers of clayNamontmorillonite crystallites are respectively shownin Fig 4b c For comparison purposes total CEDs ofvarious proportionsnumbers of the clay crystallites onthe SARSCoV2 spike and the interaction of the laterwith hACE2 are plotted in Fig Based on our experience we have hypothesized thatnanoclays due to their high adhesive properties couldalso act as SARSCoV2 inhibitors They can do it bystrongly associating with the spike S present on SARSCoV2 The results obtained from the molecularlevelsimulations of the interactions indicate that due to veryhigh CED between SARSCoV2 and the nanoclays ascompared to the former and hACE2 Fig they couldinhibit SARSCoV2 from getting engaged with hACE2Moreover it could also be concluded from Fig thatthe extent of inhibition due to nanoclays is increased inquantity dosagedependent wayNanoclay Interactions with SARSCoV2 Spike SAuthors in their earlier research have demonstrated therole of nanoclays in promoting adhesion among thecancer cells and their microenvironment and hence controlling metastasis [] Adhesion measurements of mix of Namontmorillonite and palygorskite showedan increase in adhesion by among cancer cells andthe extracellular matrix proteins Fig 6a A corresponding SEM of the nanoclays binding the Raji cells and thefibronectin proteins is shown in Fig 6b Sample imagingwas performed in SEM mode in an FEI ESEMFEG XL atthe Miller School of Medicine University ofMiami Florida Authors also discovered in their previousresearch that electrostatic van der Waals and ZP 0cAbduljauwad Nanoscale Research Letters Page of Fig Molecularlevel simulation results in Materials Studio Software a SARSCoV2 S and hACE2 CED Jcm3 b SARSCoV2 S modelinteracting with twelve crystallites of Namontmorillonite CED Jcm3 and c SARSCoV2 S model interacting with twentyfour crystallites ofNamontmorillonite CED Jcm3”obtained using Sorption technique implemented in the softwareattractions seem to be dominating in the adhesion processes [] We conclude that the same mechanismswould have also facilitated the binding of the adhesivesurfaces of the nanoclays to the spike of SARSCOV2Fig ZP is a measure of the dispersion or flocculationtendency in the colloidal form including the interactions 0cAbduljauwad Nanoscale Research Letters Page of Fig Variation of cohesive energy density CED for SARSCoV2 ShACE2 and the complexes of the former with different numbers ofNamontmorillonite crystalliteswith the other constituents present in the suspensionmedium As a general rule a zeta potential greater than mV either positive or negative indicates dispersiontendency while a zeta potential of less than mV generally results in agglomeration Higher dispersion tendencies ZP of the clay nanops used in the study ˆ’ to ˆ’ mV lead to higher dispersion tendency andhence in the generation of higher surface area amplifyingthe interactions with the SARSCoV2 spike Althoughbased on their ZP Namontmorillonite nanopshave hydrophilic nature they in the presence of saltsalso promote secondary adhesion mechanisms betweenhydrophobic and hydrophilic surfaces [] It should alsobe noted that these clay nanops have high dispersion tendency due to their hydrophilic nature and relatively higher repulsive acidbase AB interactions Table High dispersion in turn results in the generation ofhigh surface area for increasing the attractive interactions Higher surface areas promote larger attractionsdue to the van der Waal attractions and the electrostaticforces among oppositely charged surfaces Besides although of relatively lesser degree positively chargededges of Namontmorillonite ps also get electrically attracted to the spike SThe results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites Fig also confirm the above interaction behaviors It has been observed that the sorption of the claynanops results in the formation of closely interacting strong van der Waals attraction fields These van derWaals attraction fields create higher CED of the claySARSCoV2 configuration Substantial increase in totalCED after the addition of clay crystallites Fig is alsoa testimony of a very high affinity of SARSCoV2 withthese ps as compared to the affinity of the formerwith hACE2systemagglutinationNanoclays as PseudoantibodiesBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays we premise that nanoclays could be mimicked asantibodies and can thus attract and engulf coronavirusesbefore they get engaged with human hACE2 Antibodiesare glycoproteins synthesized by plasma cells as part ofthe adaptive immune response to assist in the clearanceof infection from the body Antibodies aid in infectionclearance in multiple ways such as opsonization of pathogens to facilitate phagocytosis activation of the complementandneutralization of viruses and toxins When bound to theviral surface proteins antibodies prevent the entry of theviruses into the cell by preventing the attachment of viruses to their target receptor on the cell Antibody binding can occur at different sites on the surface proteinleading to various mechanisms that cause the same effect In the case of SARSCoV2 two viral neutralizationmechanisms by antibodies have been observed [ ]and shown in Fig 1c d One of the mechanisms involvesdirect binding of antibodies to the attachment site of theSARSCoV2RBD resulting in the antibody competingwith the target receptor hACE2 Another mechanism involves the binding of antibodies to the other sites onRBD without any competition with the target receptorThe latter is shown to be involved in neutralization byof microbes 0cAbduljauwad Nanoscale Research Letters Page of Fig a Summary of adhesion force measurements among RajiRajiFN assembly using AFM before and after treatment with various proportionsof Namontmorillonite and palygorskite clay nanops [] Error bars represent the variations in the trials b SEM image of the binding of Rajicells and Fibronectin proteins produced by nanoclaysthe most potent Monoclonal Antibody mAb discoveredin the study [ ] Analogous to the antibodies interaction with SARSCoV2 RBD inhibiting the latter toengage with hACE2 a similar molecularlevel model isprepared for nanoclays resulting in a similar inhibitionof the coronaviruses and shown in Fig Owing to theirvery high affinity nanoclays would get attracted tospikes of SARSCoV2 and thus restrict engagement ofRBDs of these spikes with hACE2Proposed Nanoclay Administration MethodologyClay use as drug carriers has been tested multiple timesyielding promising results of little to no cytotoxicity tocells of the human body Kaolinite clay mineral wastested for use in a potential drug delivery system andwas shown to have high biocompatibility and very lowas[]negligiblecytotoxicity [] Poly DLlactidecoglycolidemontmorillonite nanop cytotoxicity in vitro was alsodemonstratedPalygorskitepolyethyleneiminefluorescein isothiocyanate nanocomposites also exhibited almost no cytotoxicity in vitro[] Authors have also experienced injecting nanoclayssubcutaneously for the treatment of melanoma duringin vivo studies [] Based on the use of clay as a cancerdrug carrier and in other sustainedrelease medicine[“] we propose that nanoclays may be injected asœclayalone medicine subject to the verification in vivoand clinical trialsAlthough nanoclays are nonbiodegradable a comprehensive understanding of the design of the similar inanic nanops with their metabolic performancein the body carried out in the study [] could also 0cAbduljauwad Nanoscale Research Letters Page of Fig Three possible mechanisms of interactions of montmorillonite nanoclay with the SARSCoV2 spike S Electrostatic attraction amongpositively charged nanop edges and NaCa ions with negatively charged virus surfaces Van der Waals attractions ZPelectrostatic interactionscategorize these nanoclays as human body clearable inanic agentsConclusions and RecommendationsBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays these could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2The results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites result in the formation of closely interacting strongvan der Waals attraction fields These van der Waals attraction fields create higher CED of the claySARSCoV configuration Substantial increase in total CED afterFig Interaction mechanism of nanoclay ps with SARSCoV2 spike S inhibiting the interaction of the later with hACE2 0cAbduljauwad Nanoscale Research Letters Page of addition of clay crystallites is also a testimony of a veryhigh affinity of SARSCoV2 with these ps as compared to the affinity of the former with hACE2We propose to continue the research by carrying outin vitro interaction studies between SARSCoV2 anddifferent percentage of nanoclays Based on theoptimum dose of nanoclay developed in the in vitrophase we suggest to carry out in vivo studies on the animals The animal study should be carried out both withand without nanoclay to finalize the nanoclay dose andshould lay the foundation for the clinical trialsAcknowledgementsThe authors highly acknowledge KFUPM for providing highperformancecomputing research facilitiesAuthors™ ContributionsAll the authors equally participated at all the research levels The authorsread and approved the final manuscriptFundingNo fundingAvailability of Data and MaterialsAll data generated or analyzed during this study are included in thispublished Ethics Approval and Consent to ParticipateNot applicableConsent for PublicationNot applicableCompeting InterestsThe authors declare that they have no competing interestsAuthor details1Civil Environmental Engineering department King Fahd University ofPetroleum Minerals KFUPM Dhahran Saudi Arabia 2Royal College ofSurgeons in Ireland RCSI Bahrain campus Busaiteen Bahrain 3Engineering Research International ERI Riyadh Saudi ArabiaReceived July Accepted August ReferencesEwen Callaway and Nik Spencer The race for coronavirus vaccines agraphical guide eight ways in which scientists hope to provide immunityto SARSCoV2 News Feature Nature vol April Li F Li W H Farzan M Harrison S C Structure of SARS coronavirusspike receptorbinding domain complexed with receptor Science httpsdoi101126science1116480 Li WH Angiotensinconverting enzyme is a functional receptorfor the SARS coronavirus Nature “ httpsdoi101038nature02145Li F Structural analysis of major species barriers between humansand palm civets for severe acute respiratory syndrome coronavirusinfections J Virol “ httpsdoi101128jvi0044208 Wu KL Peng GQ Wilken M Geraghty RJ Li F Mechanisms of hostreceptor adaptation by severe acute respiratory syndrome coronavirus JBiol Chem “ httpsdoi101074jbcM111325803 Wang C Li W Drabek D Okba NMA van Haperen R Osterhaus ADME A human monoclonal antibody blocking SARSCoV2 infection NatCommun Jiang S Hillyer C Du L Neutralizing antibodies against SARSCoV2and other human coronaviruses Trends Immunol “Pinto D Park YJ Beltramello M Walls AC Tortorici MA Bianchi S Crossneutralization of SARSCoV2 by a human monoclonal SARSCoV antibody Nature “da Rocha Dias S Salmonson T van ZwietenBoot B Jonsson B Marchetti SSchellens JH Pignatti F The European Medicines Agency review ofvemurafenib Zelboraf® for the treatment of adult patients with BRAF V600mutationpositive unresectable or metastatic melanoma summary of thescientific assessment of the Committee for Medicinal Products for HumanUse Eur J Cancer “Sahel N Abduljauwad and HabiburRehman Ahmed Enhancing cancer celladhesion with clay nanops for countering metastasis Nature ScientificReports April httpsdoi101038s4159801942498y Zhang Y Long M Huang P Yang H Chang S Hu Y Mao L Intercalated 2D nanoclay for emerging drug delivery in cancer therapyNano Res “ Chianelli R R Das S US Patent No Washington DC US Patent and Trademark Office Han S Liu F Wu J Zhang Y Xie Y Wu T Y Targeting of fluorescentpalygorskite polyethyleneimine nanocomposite to cancer cells Appl ClaySci “Sun B Ranganathan B Feng SS Multifunctional poly D Llactidecoglycolide montmorillonite PLGAMMT nanops decorated byTrastuzumab for targeted chemotherapy of breast cancer Biomaterials“Lin FH Lee YH Jian CH Wong JM Shieh MJ Wang CY A study ofpurified montmorillonite intercalated with 5fluorouracil as drug carrierBiomaterials “ Bothiraja C Thorat UH Pawar AP Shaikh KS Chitosan coated layeredclay montmorillonite nanocomposites modulate oral delivery of paclitaxel incolonic cancer Mater Technol 29sup3B120“B126Kevadiya BD Thumbar RP Rajput MM Rajkumar S Brambhatt H Joshi GVBajaj HC Montmorillonitepolyεcaprolactone composites asversatile layered material reservoirs for anticancer drug and controlledrelease property Eur J Pharm Sci “ Guo MY Wang AF Muhammad F Qi WX Ren H Guo YJ Zhu GS Halloysite nanotubes a multifunctional nanovehicle for anticancer drugdelivery Chin J Chem “ Martínez C D Cationic clays upon cancer therapy Virtual MultidisciplinaryConference QUAESTI Konta J Clay and man clay raw materials in the service of man ApplClay Sci “ Murray HH Traditional and new applications for kaolin smectite andpalygorskite a general overview Appl Clay Sci “ Volzone C Retention of pollutant gases comparison between clayminerals and their modified products Appl Clay Sci “ Dong Y Feng SS Poly dllactidecoglycolidemontmorillonitenanops for oral delivery of anticancer drugs Biomaterials “ Clarka KJ Sarrb AB Grantb PG Phillipsb TD Woodea GN In vitrostudies on the use of clay clay minerals and charcoal to adsorb bovinerotavirus and bovine coronavirus Vet Microbiol “ Choi G Huiyan P Alothman Z Vinu A Yun C Choy J Anionic clay asthe drug delivery vehicle tumor targeting function of layered doublehydroxidemethotrexate nanohybrid in C33A orthotopic cervical cancermodel International Journal of nanomedicine DOI httpsdoi102147IJNS95611 Hosseini F Hosseini F Jafari S M and Taheri A Bentonite nanoclaybaseddrugdelivery systems for treating melanoma Clay Minerals DOI httpsdoi101180clm201842018 Inamuddin Asiri A M and Mohammad Ali Applications of nanocompositematerials in drug delivery DOI httpsdoi101016C20160050751 Avolume in Woodhead Publishing Series in Biomaterials Zhang Y Long M Huang P Yang H Chang S Hu Y Tang A and MaoL Emerging integrated nanoclayfacilitated drug delivery system forpapillary thyroid cancer therapy doi 101038srep33335 Sci Rep Li S Xia B Javed B Hasley W D MelendezDavila A Liu M Kerzner MAgarwal S Xiao Q Torre P Bermudez J G Rahimi K Kostina N YMöller M RodriguezEmmenegger C Klein M Percec V and Good M CDirect visualization of vesicle disassembly and reassembly usingphotocleavable dendrimers elucidates cargo release mechanisms ACS 0cAbduljauwad Nanoscale Resear
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Cabozantinib is an oral multikinase inhibitor whose targets include vascular endothelial growth factor receptors MET and the TAM family of kinases TYRO3 AXL MER Cabozantinib is approved for patients with advanced hepatocellular carcinoma who have been previously treated with sorafenib based on improved overall survival and progressionfree survival relative to placebo in the phase III CELESTIAL study During CELESTIAL the most common adverse events AEs experienced by patients receiving cabozantinib included palmarplantar erythrodysesthesia fatigue gastrointestinalrelated events and hypertension These AEs can significantly impact treatment tolerability and patient quality of life However AEs can be effectively managed with supportive care and dose modifications During CELESTIAL more than half of the patients receiving cabozantinib required a dose reduction while the rate of treatment discontinuation due to AEs was low Here we review the safety profile of cabozantinib and provide guidance on the prevention and management of the more common AEs based on current evidence from the literature as well as our clinical experience We consider the specific challenges faced by clinicians in treating this patient population and discuss factors that may affect exposure and tolerability to cabozantinib IntroductionThere has been a marked increase in liver cancer deaths in recent years In there were a0 new cases of liver cancer worldwide and liver cancer accounted for almost deaths making it the sixth most prevalent cancer worldwide [] The most common primary malignancy of the liver is hepatocellular carcinoma HCC [] The frequency burden and etiology of HCC vary across geographic regions and populations but are linked to prevalence of predisposing chronic hepatic conditions such as Electronic supplementary material The online version of this s1152 contains supplementary material which is available to authorized usersKey Points Cabozantinib represents a treatment option for patients with advanced hepatocellular carcinoma who progress after sorafenibAdverse events associated with cabozantinib may be effectively managed with supportive care and dose modifications thereby allowing patients to continue treatment at the appropriate dose with minimum interruptionStudies of cabozantinib in the firstline setting are ongoing by understanding the safety profile of this drug clinicians will be able to balance efficacy with tolerability for each patient Gabriel Schwartz GabrielSchwartzucsfedu Gastrointestinal Medical Oncology Clinic University of a0California San Francisco Fourth St Fourth Floor San a0Francisco CA a0 USAIndiana University Health Simon Cancer Center Indianapolis IN USA Department of a0Medicine University of a0California San Francisco San a0Francisco CA USAIRCCS Istituto Clinico Humanitas Rozzano Milan Italy viral hepatitis and nonalcoholic fatty liver disease NAFLD or nonalcoholic steatohepatitis NASH which generally develop in the setting of cirrhosis [ ] In recent years the incidence of nonviral HCC has increased while the proportion of HCC cases related to viral hepatitis has declined [ ] Additional risk factors for HCC include alcohol consumption smoking obesity and diabetes [] As the epidemiology of these conditions has evolved so too has the etiology of HCC []Vol0123456789 0c G a0Schwartz et alFor patients with advanced HCC the vascular endothelial growth factor receptor VEGFR“targeting tyrosine kinase inhibitor TKI sorafenib has been a standard of care [] however the treatment landscape has been transformed in recent years with the introduction of newer TKIs immunotherapies and monoclonal antibody therapies [] This provides clinicians and patients with a variety of treatment options based on mechanism of action and safety profileCabozantinib is a multikinase inhibitor that targets VEGFR “ MET the TAM family of kinases TYRO3 AXL MER RET ROS1 KIT TRKB FLT3 and TIE2 [ ] several of which are implicated in tumor growth angiogenesis and immune regulation [] VEGFR MET and AXL have been implicated in the pathogenesis of HCC [“] A capsule formulation of cabozantinib was first approved in for treatment of progressive metastatic medullary thyroid carcinoma MTC [] The tablet formulation not bioequivalent or interchangeable with the capsule [] was subsequently approved for patients with advanced renal cell carcinoma RCC [ ] and more recently for patients with advanced HCC who have received prior sorafenib [ ] The approval in HCC was based on outcomes from the pivotal phase III CELESTIAL trial which showed significantly improved overall survival OS and progressionfree survival PFS with cabozantinib relative to placebo in patients who received prior sorafenib [] The safety profile of cabozantinib was manageable nearly all patients receiving cabozantinib experienced an adverse event AE but these were effectively managed with dose modification and supportive care measuresClinicians treating patients with advanced HCC can face significant challenges as many patients present with cirrhosis and comorbidities that can impact treatment tolerability Adequate assessment of liver function and management of comorbidities are therefore essential before and during HCC treatment [] Here we provide guidance on the management of AEs associated with cabozantinib in patients with advanced HCC We briefly review outcomes from CELESTIAL and focus on managing some of the more common AEs experienced by patients based on current evidence from the literature as well as our own clinical experience Cabozantinib in a0Hepatocellular Carcinoma CELESTIALIn the phase III CELESTIAL study patients with advanced HCC were randomized to treatment with cabozantinib a0mg daily or placebo [] Patients were required to have had prior treatment with sorafenib and could have received up to two prior systemic regimens for HCC Eastern Cooperative Oncology Group ECOG performance status PS of or and ChildPugh class A liver function see Electronic Supplementary Table a0 for definition were also required At the second planned interim analysis patients had been randomized The study met its primary endpoint with significantly improved OS with cabozantinib relative to placebo median OS was versus months hazard ratio confidence interval [CI] “ p a0 a0 Cabozantinib also improved PFS with a median of versus months hazard ratio CI “ p a0 a0 as well the objective response rate per Response Evaluation Criteria In Solid Tumors RECIST v11 vs a0 p a0 a0 Safety and a0TolerabilityAllcause AE rates were generally higher in the cabozantinib arm than in the placebo arm some of the more common AEs experienced by patients in the cabozantinib a0 versus placebo arms included diarrhea vs decreased appetite vs palmarplantar erythrodysesthesia PPE vs fatigue vs nausea vs hypertension vs vomiting vs asthenia vs and increased aspartate aminotransferase AST vs Fig a0 The most common grade AEs in the cabozantinib versus placebo arms were PPE vs hypertension vs increased AST vs fatigue vs and diarrhea vs Overall the safety profile of cabozantinib was consistent with those from the phase III studies in RCC and MTC with gastrointestinal GI events PPE fatigue and hypertension being the most common AEs experienced by patients across studies [ ]In addition to supportive care measures protocolspecified dose modification including dose interruption and reduction was utilized to manage AEs [] Eightyfour percent of patients in the cabozantinib arm had an AE that led to dose interruption and had a dose interruption due to a grade AE [] Sixtytwo percent of patients had at least one dose reduction due to an AE [] and dose reduced due to a grade AE [] Thirtythree percent of patients had a second dose reduction [] Median time to first and second dose reduction in the cabozantinib arm was a0days and a0days respectively PPE was the event that most commonly led to dose interruption and dose reduction followed by diarrhea and and fatigue and [] Although most patients receiving cabozantinib required a dose interruption the rate of discontinuation due to treatmentrelated AEs was relatively low in the cabozantinib arm vs in the placebo arm indicating that the majority of AEs were adequately managed with dose modification and supportive care In the cabozantinib group AEs that led to treatment discontinuation in ‰¥ a0 of patients were PPE fatigue decreased appetite diarrhea and nausea In a subgroup analysis of patients 0cAE Any grade [Grade Grade ]Fatigue [ ]Hypertension [ ]Increased AST [ ]Increased ALT [ ]Asthenia [ ]Nausea [ ]Vomiting [ ]Decrease appetite [ ]Weight loss [ ]Diarrhea [ ]Constipation [ ]Abdominal pain [ ]PPE [ ]Fig Incidence rates for select AEs experienced by patients with HCC receiving cabozantinib during the CELESTIAL trial [] AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders AE adverse event ALT alanine aminotransferase AST aspartate aminotransferase HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesiawho received sorafenib as the only prior treatment for HCC duration of prior sorafenib did not appear to impact the types or rates of grade AEs []Generally the more common AEs emerged in the first weeks of treatment Fig a0 However clinicians should be aware of infrequent or serious events that can occur in the later phases of treatment Hemorrhagic events of grade or higher were reported in of patients in the cabozantinib arm including five patients with a grade event Bleeding complications are associated with antiangiogenic therapies and may arise as a result of reduced vascular integrity [] Median time to onset of hemorrhagic events was a0weeks in CELESTIAL Other grade or higher rare but serious AEs in patients receiving cabozantinib included fistulas of patients GI perforations and arterial and venous or mixed thrombotic events [] Median time to first occurrence was approximately a0weeks for GI perforations weeks for venous and arterial thromboembolisms and weeks for fistulas [] Two patients in the cabozantinib arm had developed ChildPugh C ie decompensated cirrhosis by the week assessment []Reversible posterior leukoencephalopathy syndrome RPLS a syndrome of subcortical vasogenic edema diagnosed by magnetic resonance imaging has been reported with cabozantinib and other TKIs [ ] Although there were no RPLS events during CELESTIAL [] clinicians should be aware of the symptoms which include headaches seizures confusion changes to vision or altered mental function [ ] Osteonecrosis of the jaw ONJ whereby necrotic jaw bone becomes exposed is another rare but serious AE associated with TKIs including cabozantinib [“] although again there were no ONJ events reported during this study [] The use of antiresorptive drugs in patients with bone metastases is also associated with development of ONJ []A post hoc analysis estimated the incremental qualityadjusted lifeyears accrued with cabozantinib compared with placebo using the fivedimension fivelevel EuroQol questionnaire [] Cabozantinib treatment was associated with an initial decline in mean total qualityadjusted lifeyears during the first “ a0months relative to placebo followed by longterm improvement that was significantly greater than that observed with placebo p a0 a0Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c Fig Rates and timing of select AEs in patients with HCC receiving cabozantinib during the CELESTIAL trial The size of the circle is proportional to the AE rate AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders black generalother AE adverse event ATE arterial thrombotic event GI gastrointestinal GR grade HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesia VTE venous thrombotic eventMedian time to first dosereduction to mg weeksG a0Schwartz et alMedian time to seconddose reduction to mg weeksFistulas Hemorrhage GR ATEs VTE Wound complication GI perforations Hepatic encephalopathy Diarrhea PPE Hypertension Median time to first occurrence weeks Factors Affecting Tolerability of a0Cabozantinib Co‘morbiditiesHCC emerges primarily in older adults [] In addition to the underlying HCC etiology older adults with HCC are likely to have additional comorbidities such as cardiovascular or pulmonary disease [] and it is not uncommon for patients with HCC to have multiple comorbidities [] Liver cirrhosis with compromised liver function and decreased hepatic reserve is a major risk factor for HCC development Other HCCrelated comorbidities include hepatitis B virushepatitis C virus infection alcoholic liver disease NASH and diabetes [] In addition metabolic syndrome characterized by hyperlipidemia and hypertension is linked to development of NAFLD which may progress to NASH cirrhosis and finally HCC [] For patients with HCC assessment of liver function is a key step in treatment decisionmaking [] Patients with moderate or severe hepatic impairment are predominantly excluded from clinical trials in HCC therefore treatment of these patients is complicated by a lack of prospective clinical data as well as competing comorbidities []Although the number of patients with HCC and prior an transplant is limited these patients are generally excluded from clinical trials and treatment is complicated by the need for immunosuppression TKIs may be used to treat posttransplant HCC recurrence although supporting data are limited The use of TKIs in these patients is complex so treatment decisions should involve collaboration between the oncology and transplant medicine care teams The use of sorafenib in patients receiving mammalian target of rapamycin inhibitorbased immunosuppression has been associated with an increased risk of fatal bleeding [ ] Immunotherapies are associated with an increased risk of an rejection in posttransplant patients [] Cabozantinib Clearance and a0ExposureTKIs are associated with high interpatient variability in clearance and exposure which may affect both efficacy and tolerability This variability may be due to a variety of factors including genetic background drug“drug interactions drugfood interactions and renal or hepatic impairment [] As evidenced by exposureresponse modeling patients with low clearance of cabozantinib may have higher exposure and an increased risk of developing certain AEs [ ] Awareness of these nuances may help clinicians to mitigate their effects thereby balancing efficacy with tolerability Hepatic and a0Renal ImpairmentAccording to pharmacokinetic analyses of patients with HCC and other tumor types mild hepatic impairment is predicted to have a minimal effect on cabozantinib exposure [] therefore adjustment of the recommended 60mg starting dose is not necessary for patients with Child“Pugh A 0cliver function [ ] Data on the pharmacokinetics of cabozantinib in patients with moderate ChildPugh B or severe Child“Pugh C hepatic impairment are limited [] As per the US Food and Drug Administration FDA prescribing information the starting dose of cabozantinib should be reduced to mg in patients with moderate hepatic impairment while cabozantinib is not recommended for patients with severe hepatic impairment [] Note that the European Summary of Product Characteristics SmPC does not recommend dose adjustments for moderate hepatic impairment owing to limited data [] For patients with HCC increased exposure due to hepatic impairment should be considered if intolerable AEs develop and dose modification undertaken as recommended Fig a0 [ ] Cabozantinib should be used with caution in patients with mild or moderate renal impairment owing to the potential for increased exposure although no dose adjustments are necessary Cabozantinib is not recommended for use in patients with severe renal impairment owing to lack of data on safety and efficacy in this population [ ] Drug“Drug and a0Drug“Food InteractionsGiven the range of comorbidities that may exist in patients with advanced HCC it is important to review all concomitant medications for potential interactions prior to initiation of treatment with cabozantinib Certain medications and foods have been shown to modulate the pharmacokinetics of cabozantinib which may in turn impact exposure levels efficacy and risk of AEs Cabozantinib is metabolized in the liver primarily by the enzyme cytochrome P450 3A4 CYP3A4 [] therefore CYP3A4 inhibitors or inducers may impact exposure examples of CYP3A4 inducersinhibitors are shown in Electronic Supplementary Table a0 Strong CYP3A4 inhibitorsinducers should be avoided in patients receiving cabozantinib If concomitant administration of a strong CYP3A4 inhibitor is necessary then the cabozantinib Recommended dose at initiation mg Except for¢ Patients with moderate hepatic impairment or coadministration of a strong CYP3A4 inhibitor initiate cabozantinib at mg ¢ Patients with coadministration of a strong CYP3A4 inducer initiate cabozantinib at mg Safety assessmentNo AEsGrade Grade AE or ONJSupportive caresee Tables “DosemodificationImprovementtolerableelbarelotnIlitnu esod dloHgrade ‰¤Continue at tolerated doseReduce dose by mg and restart mg †’ mg mg †’ mg mg †’ mg mg †’ mg or discontinueImmediate Discontinuation¢ Severe hemorrhage¢ Development of GI perforation or unmanageable fistula¢ Serious thromboembolic event eg myocardial infarction cerebral infarction¢ Hypertensive crisis or severe hypertension despite optimal medical management¢ Nephrotic syndrome¢ Reversible posterior leukoencephalopathy syndromeFig Cabozantinib dosing algorithm [ ] AE adverse event CYP3A4 cytochrome P450 3A4 GI gastrointestinal ONJ osteonecrosis of the jawManagement of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et aldose should be reduced by a0mg for example from to a0mg [] Conversely the cabozantinib dose should be increased by a0mg if strong CYP3A4 inducers need to be coadministered [ ]Cabozantinib should not be taken with any food as this may affect absorption [] The label recommends that cabozantinib be taken at least h before or at least h after eating [] Grapefruit and grapefruit juice are strong CYP3A4 inhibitors and should be avoided [ ]Cabozantinib may be used with caution in patients who are receiving concurrent antiarrhythmics or other QTprolonging agents [] This is based on a study of patients with MTC who received a daily 140mg capsule dose of cabozantinib recommended for this indication in which the mean deltadelta QT interval was increased by approximately “ a0ms with upper CIs not exceeding ms [] Such an increase is within the range considered to be acceptable for oncology drugs in this setting [] No patient in the aforementioned study or in CELESTIAL had a confirmed QTcF QT corrected using Fridericia™s method a0 ms [] which is considered clinically significant [] For patients receiving cabozantinib monitoring with periodic electrocardiogram and electrolyte measurements may be advisable particularly in patients with risk factors such as cardiac disease or a prior history of QT prolongation [] Concomitant use of proton pump inhibitors PPIs such as esomeprazole does not affect cabozantinib exposure levels [] However PPIs may cause hypomagnesemia which is linked to an increased risk of QT prolongation [] Therefore coadministration of PPIs and cabozantinib should be undertaken with caution following an individualized assessment of the patient™s baseline magnesium levels and concomitant medications that may also influence QT Pretreatment AssessmentsGiven the heterogeneity of the HCC patient population and the complexity associated with comorbidities and concomitant medications all patients should undergo a comprehensive assessment of medical history prior to initiation of treatment with cabozantinib Ideally the multidisciplinary care team should include an oncology pharmacist [] A œbrown bag medication review should be carried out prior to treatment initiation [] whereby the patient brings in all current medications including overthecounter medicines vitamins herbal remedies etc Therapeutic duplications should be eliminated for example concomitant PPIs and histamine H2 antagonists H2 blockers Switching and deprescribing should be considered where possible to minimize the risk of drugdrug interactions Adverse Event ManagementThe AE profile of cabozantinib is generally similar to that of other VEGFRtargeting TKIs with GIrelated AEs fatigue PPE and hypertension being the most common AEs [] Other AEs that occur less frequently can also have a significant impact on quality of life QoL and treatment adherence such as mucosal inflammation [] Hepatobiliary AEs such as elevated AST alanine aminotransferase ALT and bilirubin are particularly relevant in the context of advanced HCC and need to be carefully monitoredProphylactic and supportive care measures for the more common cabozantinibassociated AEs grade or tolerable grade are outlined in Tables a0 and discussed in the upcoming sections Symptom gradings are summarized in Electronic Supplementary Table a0 Dose interruption is recommended for management of intolerable grade AEs not resolved with supportive care measures or for any grade AEs Fig a0 [ ] Cabozantinib may be reinitiated at a reduced dose once the event resolves to grade ‰¤ a0 Palmar“Plantar ErythrodysesthesiaPPE is one of the more common events associated with anticancer therapies including VEGFRtargeting multikinase inhibitors [“] PPE is characterized by pain redness tingling and swelling of hands and feet [] Presentation may vary according to the etiologic agent PPE induced by TKIs is typically localized to pressurebearing areas in contrast to that caused by chemotherapy which has a more diffuse pattern It has been hypothesized that inhibition of multiple angiogenic pathways by TKIs may compromise repair of capillary microtrauma in areas exposed to mechanical stress such as the hands and feet [ ] Although not lifethreatening PPE can rapidly progress to a debilitating condition negatively impacting QoL [ ]Prophylaxis and prompt management of emerging symptoms may help to minimize the impact of PPE on QoL and adherence Table a0 Prophylactic measures predominantly involve skin care practices to remove hyperkeratotic areas and to minimize friction and damage prior to the start of treatment [ ] Recommendations include use of thick cotton gloves and socks padded insoles in shoes and avoidance of heat or friction on the hands and feet [ ] Patients with potentially predisposing comorbidities such as peripheral neuropathy [ ] as well as patients with persistent symptoms may benefit from involvement of a podiatrist andor dermatologist within their multidisciplinary care team [] Treatment strategies involve moisturization prevention of infection and analgesia [ ] Monitoring is crucial so that emerging symptoms can be proactively managed Patients should be assessed at baseline 0cTable Adverse event management strategies”palmar“plantar erythrodysesthesia PPE PPEProphylaxisProvide education on prophylactic skin care before starting treatment []Advise manicure and pedicure before and during treatment to remove hyperkeratotic areas [ ]Protect sensitive areas recommend sunscreen with SPF protection ‰¥ a0 thick cotton gloves and socks padded insoles and wellfitting shoes avoid heat sources and use cooling aids and avoid activities that may cause force or rubbing on the hands and feet eg heavy lifting dish washing [ ] delegate such tasks to caregiversAdvise on optimal hand cleaning avoid fragrancedfoaming soaps and hand sanitizers containing alcohol ensure hands are dried thoroughly after cleaning []Prophylactically administer keratolytic cream eg urea [ ]Monitor regularly in order to proactively manage skin toxicities evaluate at baseline monitor up to weekly for the first “ months and monthly thereafter [ ]Supportive careContinue prophylactic measures []Maintain moisture of skin using emollients [ ]Consider topical treatment with salicylic acid urea “ cream either alone or with tazarotene cream or fluorouracil cream andor clobetasol cream topical analgesics may be added for pain control [ ]Topical cortisone and clobetasol may also be used consider oral analgesics eg NSAIDs pregabalin cautious use of opioids [ ]Consult with a dermatologist to drain blisters and remove hyperkeratotic areas []To prevent infection of cracked skin soak in equal parts vinegar and water for min per day [] a0Antibiotics should be prescribed only if there is evidence of infection [] a0There is limited evidence for the use of pyridoxine vitamin B6 []NSAID nonsteroidal antiinflammatory drug SPF sun protection factorTable Adverse event management strategies”fatigue FatigueProphylaxisProvide patient education about fatigue management tools and available support []Establish baseline fatigue levels with a fatigue scale and remeasure regularly during patient visits []Ensure adequate fluid and nutritional intake []Advise behavioral modifications balancing rest with physical activity recommendations include relaxation massage yoga aerobic or resistance exercise programs and energy conservation strategies [“]Assess thyroid function prior to treatment and monitor during treatment [ ]Supportive careRule out alternative causes of fatigue eg anemia endocrine disorders such as hypothyroidism pain dehydration hypercalcemia or depressionanxiety [ ]Advise patient to increase activity consider referral to a physical therapist []Consider referral to nutritional counselor for nutritional therapy []Incorporate psychosocial measures including cognitive therapy social support biofeedback and sleep therapy []Incorporate management with psychostimulants eg methylphenidate [ ] or corticosteroids eg methylprednisolone []Owing to effects on CYP3A45 substrates including cabozantinib longterm use of modafinil should be avoided []CYP3A4 cytochrome P450 3A4 CYP2C19 cytochrome P450 2C19Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et alTable Adverse event management strategies”gastrointestinal GastrointestinalDiarrheaProphylaxisInstruct patients to monitor food and fluid intake [] a0Recommended water intake per day from all beverages and food [] L oz for women L oz for men a0Advise patients to keep a stool diary and to promptly report diarrhea to their healthcare provider [ ]Advise patients to avoid foods that may cause GI events such as lactosecontaining foods caffeine highfat or highfiber food eg nuts seeds legumes and raw fruit and vegetables [ ]Implement dehydration prevention management through oral rehydration with electrolytes []Supportive careAdminister loperamide at the first sign of diarrhea [ ] a0 mg orally followed by mg every h until “ h after last bowel movement maximum of mg in h a0For chronic diarrhea “ mg twice daily titrated as needed a0Alternatives to loperamide include diphenoxylate and tincture of opium []Implement supportive dietary modifications continuous oral hydration correction of fluid and electrolytes small frequent meals avoid lactosecontaining food and drink [ ] a0The BRAT bananas rice applesauce toast diet may help to alleviate mild diarrhea []If there are signs of severe dehydration administer IV fluid replacement isotonic saline or balanced salt solution []Rule out nontreatmentrelated causes eg infectious diarrhea []Decreased appetiteProphylaxisAdvise patients to monitor their appetite and weight []Encourage patients to consume highprotein calorierich food fruit and vegetables nutritional supplements that they may snack on throughout the day [ ]Advise patients to preprepare and freeze nutritional preferred food []Supportive careTreat underlying nausea []Consider involving a dietitian who may recommend scheduled eating times []Recommend a highcalorie diet []Provide dietary education alongside dietary modifications andor nutritionalvitamin supplements []Use a pharmacologic agent to stimulate appetite such as a CB1 receptor agonist dronabinol [ ] systemic corticosteroid methylprednisolone [ ] progestin megestrol acetate [ ] or mirtazapine [ ]NauseavomitingProphylaxisAssess risk factors for nauseavomiting prior to treatment []Metoclopramide may be administered prophylactically []Advise patients to avoid foods that are overly sweet greasy fried or spicy []Supportive careAntiemetic agents such as dopamine receptor antagonists eg metoclopramide prochlorperazine or 5HT3 receptor agonists eg ondansetron are recommended for management of nausea or vomiting [ ] a0Certain NK1 receptor agonists eg aprepitant and netupitant and dexamethasone are inducers inhibitors andor substrates of CYP3A4 and thus could alter cabozantinib exposure [ ] however the potential for ondansetron to prolong the QT interval must also be considered [] There is moderate evidence for olanzapine an antipsychotic drug that blocks multiple neurotransmitters as an antiemetic in this setting [] 0cTable continuedMucosal inflammationstomatitisProphylaxisA comprehensive dental examination should be conducted prior to treatment to identify potential complications []Mitigation of potential risk factors [ ] a0Modification of illfitting dentures a0Appropriate care for preexisting dental problems such as caries ulcers etcRegular oral assessments should be conducted throughout treatment [ ]Educate patients on good oral hygiene and oral care protocols including written instructions [] a0The oral cavity should be washed using salinecontaining mouthwash up to four times daily and dentures should be regularly cleaned []Painful stimuli eg smoking alcohol hot fooddrink sharp or spicy food should be avoided [ ]Supportive careTreat pain with doxepin mouthwash or viscous lidocaine [ ]Lactobacillus lozenges may be used to reduce inflammation []Obtain bacterialviral culture if oral infection is suspected and treat infection as clinically indicated []5HT3 5hydroxytryptamine CB1 cannabinoid CYP3A4 cytochrome P450 3A4 GI gastrointestinal IV intravenous NK neurokininTable Adverse event management strategies”hypertension HypertensionProphylaxisMonitor BP before initiation of cabozantinib using a minimum of two standardized BP measurements alongside patient history physical assessment directed laboratory evaluation and an instrument test to determine cardiovascular risk factors [ ]Educate patients on BP selfmonitoring and advise they keep a BP log []BP should be well controlled prior to initiating cabozantinib ensure patients who have already been prescribed antihypertensive therapy are adherent and that therapy has been titrated to effective doses [ ]Check for potential drugdrug interactions of existing antihypertensive agents with cabozantinib Supplementary Table a0Consider effects of concomitant medications on BP eg antiinflammatory drugs can increase BP opiates can lower BP []Monitor BP during cabozantinib treatment weekly during first cycle every ‰¥ a0“ weeks thereafter []Supportive careAdd antihypertensive medications or increase dose of existing medication as indicated [ ]Patients with portal hypertension should be treated with nonselective betablockers []The antihypertensive agent should be carefully considered owing to potential inhibition of CYP3A4 [ ] Supplementary Table a0 a0Thiazides angiotensinconverting enzyme inhibitors and angiotensin receptor blockers may be used to treat hypertension and are not known CYP3A4 substrates [“ ] a0Thiazide diuretics should be prescribed with caution owing to the associated risk of diarrhea [] a0Diltiazem and verapamil are moderate inhibitors of CYP3A4 [] a0Amlodipine felodipine lercanidipine nisoldipine and nifedipine are not considered to be CYP3A4 inhibitors []BP blood pressure CYP3A4 cytochrome P450 3A4and monitored at least weekly for the first “ months of treatment and monthly thereafter [ ] Close monitoring in the early stages of treatment need not involve weekly visits”phone calls from a clinician nurse or pharmacist may facilitate monitoring in between scheduled appointments [] Patients should be encouraged to report early signs of PPE to their healthcare provider [] it may also be reassuring for patients to know that early reporting and management of AEs
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" recently copy number alteration cna of 9p241 were demonstrated in of diffuse large bcelllymphoma dlbcl with gene expression and mutation profiles that were similar to those of primary mediastinallarge bcell lymphoma pmbcl however their cnabased profile and clinical impact still remain unclearmethods multiplex ligationdependent probe amplification were employed to investigate the prevalence of jak2pdl2 amplification in dlbcl and their cnabased pattern of driver genes the clinical outcome and characteristicswere also analyzedresults using unsupervised hierarchical clustering a small group of dlbcl was clustered togetherwith pmbcl as cluster_2 demonstrating amplification of jak2 and pdl2 this subgroups ofdlbcl demonstrated significant higher expression of pdl1 than those with myd88 l265p mutationp andthey exhibited dismal os and pfs as compared with dlbcl_othersp and respectively which issimilar to dlbcl with myd88 l265p mutations dlbcl with amplification of jak2pdl2 exhibits cna pattern that is similar to pmbcl anddemonstrates unfavorable clinical outcome that resembles those with myd88 l265p mutation it is essential toidentify this subgroup of dlbcl who may acquire more benefits from the jak2 and pdl1 signaling inhibitionkeywords diffuse large bcell lymphoma jak2 pdl2 amplification prognosis diffuse large bcell lymphoma dlbcl is a highly heterogeneous disease recently several distinctive geneticsubtypes were identified including schmitz r studymcd bn2 n1 and ezb subtypes and chapuy b study c0 c5 clusters [ ] godfrey j study also correspondence jmyingcicamsaccn lvningcicamsaccn xuemin xue and wenting huang are cofirst authors jianming ying andning lv are cosenior authors1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing chinafull list of author information is available at the end of the identified an unique biological subset of dlbcl withpdl1 gene alterations having high risk features thus the genetics of dlbcl relating to potential therapeutic targets for immune checkpoint inhibitors shouldbe paid much more attention tojanus kinase jak2 programmed cell death ligand pdl1cd274pdcd1lg1 and programmed celldeath ligand pdl2cd273pdcd1lg2 are adjacent to each other on chromosome 9p241 playing keyroles in host immune surveillance amplification of9p241 were frequently seen in celllines of classical and primaryhodgkin lymphoma chl 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 0cxue bmc cancer page of mediastinal large bcell lymphoma pmbcl but much less in dlbcl cell lines ] correspondingly pd1 ligands pdl1 and pdl2transcripts and proteins were more abundant in chl andpmbcl cell lines than that in dlbcl cell lines recently y wang study demonstrated that ofdlbcl had copy number alteration cna of 9p241with a gene expression and mutation profile similar tothose of pmbcl however their cnabased profileand clinical impact still remain unclearin thisthereforestudy we employed multiplexligationdependent probe amplification mlpa to investigate the prevalence of jak2pdl2 amplification indlbcl and their cnabased pattern of driver genesincluding bcl2 cdkn2a and tp53 and we analyzed their longterm survival outcome after treatmentof rchoplike regimemethodscase selectionwe collected consecutive cases of dlbcl and pmbclin our clinical ffpe archives of excisional biopsy database between jan and oct and cases ofdlbcl and cases of pmbcl were found after confirmation one case of dlbcl was diagnosed as pmbclthus cases of dlbcl and cases of pmbcl wereacquired finally see additional file all patients werediagnosed at national cancer centernational clinicalresearch center for cancercancer hospital chineseacademy of medical sciences and peking union medicalcollege according to the revised 4th edition ofthewho classification of tumours of haematopoietic andlymphoid tissues the data regarding treatment andprognosis were acquired by means of medical recordconsultation and telephone conversationmultiplex ligationdependent probe amplification mlpagenomic dna were extracted from formalinfixedparaffinembedded ffpe blocks using qiaamp dnaffpe tissue kit qiagen valencia ca then dnacopy number quantification and myd88 l265p mutation were detected using mlpa kitmrchollandnetherlands the pcr products were detected on anabi genetic analyzer applied biosystems usaand the final result were analyzed using coffalyser software the relative peak ratio prr of probe largerthan was defined as amplification and less than was defined as deletion see additional file geneswhich had two or more probes covering two differentexomes were put into final analysis including jak2 pdl2 mdm2 rel pus10 bcl2 nfatc1 spib foxp1nfkbiz bcl6 prdm1 tnfaip3 cdkn2a ptening1 and tp53 the details of mlpa probes of drivergenes in dlbcl are shown in the online supportingmaterial see additional file true amplification of onegene was regarded only when all probes of this gene exhibited amplification and vice versa see additional file myd88 l265p mutation was identified when theprobe had a high peak myd88 wildtype didn™t show anypeak see additional file immunohistochemistry ihc staining of pdl122c3ihc staining was performed on dako autostainer link asl48 platform each ffpe block were cut at athickness of 4μm and then deparaffinized antigen retrieval were performed using the envision„¢ flex targetretrieval solution at low ph monoclonal pdl1clone 22c3 dako were used as primary antibodyfollowed by incubation with envision„¢ flex mouselinker and then envision„¢ flex hrp reagent finally the ihc was visualized by envision„¢ flex dabeach ihc slide contained a positive controllungcarcinomaihc score of pdl1 were calculated by multiplyingthe percentage of positive cells with mean intensity no staining weak staining moderate staining strong staining which was reported in previous study the results were evaluated by an experienced hematopathologist xueminstatistical analysisthe differences of clinicopathological characteristicsamong different groups were analyzed using chisquaretest fisher exact test or kruskalwallis rank sum testpdl1 ihc score between different groups was analyzedusing wilcoxon test overall survival os and progressfree survival pfs times were defined from the date ofpathologic diagnosis to the date of the event or the lastfollowup the hazard ratio hr of each parameter wascalculated by univariate cox proportional regressionanalysis firstly in which parameters with p wereevaluated together using multivariate cox proportionalregression analysis the survival curve were made according to kaplan“meier procedure the day oflastfollowup was march 1st all statistical analysiswere two sided and p was defined as significanceunsupervised hierarchical clustering was carried outusing euclidean distance and complete method heatmap was plot using pheatmap packageall above statistical analyses were run in r statistic softwareresultsunsupervised hierarchical clustering of cnas of drivergenes and its survival analysis in dlbcl and pmbclpatientsbased on array cgh lenz g study previouslyidentified specific cnas in pmbcl which were different 0cxue bmc cancer page of from abc and gcb of dlbcl abc dlbcls oftenhave cnas in foxp1 nfkbiz cdkn2a cdkn2binf4a bcl2 nfatc1 and spib while gcb dlbclsfrequently harbor cnas in rel pten mdm2 mihg1and ing1 pmbcl often demonstrate cnas of jak2 andpdl2 using unsupervised hierarchical clustering we explored the cnabased pattern of these genes in dlbcl andpmbcl the result showed that a small group of dlbcl was clustered together with pmbcl as cluster_2 with amplification of jak2 and pdl275068fig 1a this subgroup of dlbcl occurred atthe site of cervical lymph node cases gastrointestinal tract cases nasal cavity case and spleen cases fig 1atable 1additional file the frequency of jak2 and pdl2 amplification in the whole cohort of dlbcl were and while both of them were inpmbcl fig 1a see additional file meanwhile all casesin cluster_3 harbored amplification of nfkbiz which is essential for nfκb activation in abc dlbcl but noamplification of nfkbiz was found in cluster_1as to survival dlbcl in cluster_2 demonstrated significant worse os p and pfs p as compared with dlbcl in cluster_1fig 1b howevercluster_1 and cluster_3 didn™t reveal significant differencein survival fig 1b we also analyzed the os and pfs between dlbcl with and without jak2pdl2 amplification and got statistical significance see additional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table fig heatmap and survival analysis based on unsupervised hierarchical clustering and status of jak2pdl1 amplification and myd88 mutationin tcga dataset a heatmap of cnabased profiles of driver genes in dlbcl and pmbcl by using unsupervised hierarchical clustering b survivalcurves and coxregression analysis of os and pfs among three cnabased clusters after rchoplike treatment c status of amplifications of jak2pdl1cd274 and pdl2pdcd1lg2 and mutation of myd88 in dlbcl tcga pancancer atlas from cbioportal [ ] 0ccervical lymphnodefemale high_intermediatehigh_intermediatedlbclnasal cavitymalegcbbreak_apartdlbcldlbcldlbcldlbcldlbcldlbclcervical lymphnodestomachstomachcolondlbclpmbclpmbclpmbclpmbclcervical lymphnodecervical lymphnodemediastinummediastinummediastinummalelow_intermediate non_gcbnormalfemale low_intermediate non_gcbmalemalelowhighnon_gcbgcbnon_gcbnormalnormalnormalnormalfemale low_intermediate non_gcbnormalmalelowfemale female female lowlowhigh_intermediatenanananananormalnormalnormalnormalnormaljak2_amppdl2_amp“““““““““““““xue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265p“myc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didn™t show any significant differences table jak2pdl2 amplification identify a distinctive cnabasedpattern of dlbcl similar to that of pmbclsince dlbcl with jak2pdl2 amplification had less frequency of myd88 l265p mutation our study separateddlbcl patients into three subgroups dlbcl with jak2pdl2 amplification dlbcl_jak2pdl2_amp dlbclwith myd88 l265p mutation dlbcl_myd88_l265pjak2pdl2 amplification norand dlbcl withoutmyd88_l265p mutation dlbcl_others fig 2a basedon the unsupervised cluster result fig 1a one patientwho had both jak2pdl2 amplification and myd88l265p mutation was clustered into cluster_2 thereforethis patient was put into dlbcl_jak2pdl2_amp subgroup accordingly we also analyzed the data when thiscase was included in dlbcl_myd88_l265p subgroupand got the similar result see additional file unlike dlbcl_myd88_l265p and dlbcl_othersdlbcl_jak2pdl2_amp showed a distinctive pattern similar to that of pmbcl with high frequencyof rel and nfkbiz amplifications but no amplification of bcl2 and nfatc1 and no deletion ofprdm1 was found fig 2awith respectto clinicopathologicdlbcl_jak2pdl2_amp tend to be youngerdlbcl_myd88_l265p p hans modelcharacteristicsthantable whileinternational prognostic index ipi riskpdl1 expression in dlbcl with jak2pdl2 amplificationwas significantly higher than that in dlbcl with myd88l265p mutationtotally cases were performed pdl1 22c3 ihc detection including dlbcl_myd88_l265p cases dlbcl_jak2pdl2_amp cases dlbcl_others cases andpmbcl cases the result showed that pdl1 expressionin dlbcl_jak2pdl2_amp was significantly higher thanthat in dlbcl_myd88_l265p p and dlbcl_others p fig 2b and d while no significant difference was found between dlbcl_jak2pdl2_amp andpmbcl p fig 2bjak2pdl2 amplification identify a subgroup of dlbclwith unfavorable survival outcome similar to that ofmyd88 l265p mutationtrying to explore the survival indication of jak2pdl2 amplification and myd88 l265p mutation cases of dlbcls who received rchoplike regimentwith or without surgical resection were enrolled toperformed cox proportional regression analysis of osand pfs the median followup time was monthsrange “ monthsin the univariatecompared withdlbcl_others dlbcls with myd88 l265p mutationhad significantly worse os and pfs p andanalysisas 0cxue bmc cancer page of fig comparison of cnabased pattern pdl1 expression and survival analysis among pmbcl and three subgroups of dlbcl a comparison ofcnabased patterns of driver genes among pmbcl and three subgroups of dlbcl according to the status of jak2pdl2 amplification andmyd88 l265p mutation b comparison of pdl1 expression ihc score among pmbcl and three subgroups of dlbcl c survival curves and coxregression analysis of os and pfs among three subgroups of dlbcl after rchoplike treatment d representative images of he× and pdl1× ihc in dlbcl_jak2pdl2_amp and dlbcl_ myd88_l265p respectively and the same to dlbcls withjak2pdl2 amplification p and respectively meanwhile ipi risk category were significantly associated with os and pfs fig 2c tables and in the multivariate analysis ipi risk category andthree subgroups of dlbcl were put into analysis ascompared with dlbcl_others dlbcl with myd88l265p mutation still showed poor os and pfs p and respectively and the same todlbcl with jak2pdl2 amplification for pfs andos p and respectively meanwhile ipirisk category was still an independent risk predictorsfor os and pfs fig 2c tables and either jak2pdl2 amplification or myd88 l265pmutation are frequently seen in relapserefractory dlbclwith pfs less than yearsdlbcl with pfs less than years was defined as primaryrelapserefractory cases among these cases who treated byrchoplike regime the frequency of jak2 and pdl2amplification were and meanwhilethe frequency of myd88 l265p mutation were dlbcl with either jak2pdl2 amplification ormyd88 l265p accounted for discussiondlbcl presents with a wide spectrum of genetic aberration recently shi study exhibited pdl2 amplification in pmbcl and of dlbcl chapuy demonstrated of 9p241 amplification indlbcl meanwhile dlbcl with pdl1 gene alterations was identified as a unique biological subgrouphaving high risk features y wang study demonstrated that of dlbcl had cna of 9p241 withgene expression and mutation profiles that were similarto those of pmbcl in our study by using unsupervised hierarchical clustering cases ofdlbcl were clustered together with pmbcl as cluster_2 indicating that they shared recurrent cnas theywere enriched for jak2 amplification and pdl2 amplification fig 1a 0cxue bmc cancer page of table comparison of characteristics among pmbl and three subgroups of dlbcldlbclothersmyd88_l265pjak2pdl2_amppmbclpatientsage median range “ “ “ “bmnegativepositiveihc hans™ algorithmgcbnongcbipilowrisklow_intermediatehigh_intermediatehighmyc breakapartnegativepositive p_valueχ2test kruskalwallis rank sum testusing hans model most of dlbcl in cluster_2 werenongcb and tend to be younger than othergroups of dlbcl table which was consistent withprior study therefore coupled with y wang study we confirmed that dlbcl with jak2pdl2 amplification is a unique subgroup resembling the pmbclwith respect to cna patternwith regard to survival increasingly data exhibited thatthe suppression of immune surveillance in dlbcl was associated with poor survival godfrey j study hasdemonstrated that dlbcl with pdl1 gene alterationsshowed high risk features metaanalysis also showedthat pdl1 expression was associated with poor os andadverse clinicopathologic features in dlbcl in y wang study of dlbcl harbored cnaof 9p241 of which were gains and were amplifications and as compared with those who have nogain of 9p241 dlbcl with 9p24 amplification had atrend of better efs while patients with only gain tend tothey didn™thave worse prognosis unfortunatelyshow any statistical significance in our study of dlbcl were found that had cna of jak2when jak2 cna was separated into gain mlpa valuebetween “ and amplification mlpa value as described cases in dlbcl_jak2pdl2_amp group were found that had jak2 gain whichwas slightly lower than that in wang j study asshown in additional file and both dlbcl withjak2 gain and with amplification demonstrated significant poor prognosis as compared with rest of dlbclas shown in additional file more interesting unlikey wang study cases of pmbcl were included in our study as control all of which demonstrating jak2 gains rather than amplifications as shown inadditional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table which was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1cmyd88 l265p is a poor indicator of survival for dlbcl which may lead to primary refractoryrelapsed diseasethis is a gainoffunction driver mutation occurring in of dlbcl but absent in pmbcl [“] inour study the frequency of myd88 l265p in dlbcl andpmbcl were and which were in linewith prior studies [“] of greatinterest myd88l265p mutation occurred less frequently in cluster_2 which was supported by the data tcga pancancer atlas from cbioportal [ ] thus when we divided dlbcl patients into three subgroups dlbcl_jak2pdl2_amp dlbcl_myd88_l265p and dlbcl_others both dlbcl_jak2pdl2_amp and dlbcl_myd88_l265p demonstrated dismal os and pfs with amedian followup of years as compared with dlbcl_others therefore dlbcl with jak2pdl2 amplification 0cxue bmc cancer page of table os in dlbcl treated by rchoplike regimeage ‰¥ bmnegativepositivesiteextranodalnodalihc hans™ algorithmgcbnongcbmyc fish breakapartnegativepositiveipi risk categorylowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clustercluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_jak2pdl2_ampdlbcl_myd88_l265poshr_u95ci “ “ “ “ “ “ “ “ “ “ “ “ “p_valueoshr_m95cip_value “ “ “ “ “hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasn™t put into multivariate analysiswas identified as a poor survival subgroup that is similar todlbcl with myd88 l265p mutationmeanwhile we also compared the cna patterns ofdriver genes among dlbcl_jak2pdl2_amp dlbcl_myd88_l265p dlbcl_others and pmbcl dlbcl_jak2pdl2_amp showed a distinctive pattern similarto pmbcl with high frequency of rel and nfkbizamplifications but no amplification of bcl2 and nfatc1 and no deletion of prdm1 was found the profile ofdlbcl_myd88_l265p was closed to dlbcl_othersshowing relatively high frequency of cdkn2a deletionnfatc1 amplification and bcl2 amplificationin our study of dlbcl_jak2pdl2_ampharbored both jak2 and pdl2 amplifications simultaneouslyindicating that they may also have the pdl1amplification because pdl1 located in the middle ofjak2 and pdl2 at 9p241 thus we hypothesized thatpdl1 expression would be upregulated in this subgroup as what we expected using pdl1 22c3 ihcdetection pdl1 expression in dlbcl_jak2pdl2_in dlbcl_amp was significantly higher than thatmyd88_l265p p and dlbcl_othersp fig 2b and d but not in pmbcl p fig 2b meanwhile pdl1 expression could be 0cxue bmc cancer page of table pfs in dlbcl treated by rchoplike regimeage ‰¥ bmnegativepositivesiteextranodalnodalihc hans™ algorithmgcbnongcbmyc breakapartnegativepositiveipilowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clusterscluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_ jak2pdl2_ampdlbcl_ myd88_l265ppfshr_u95ci “ “ “ “ “ “ “ “ “ “ “ “ “p_value pfshr_m95cip_value “ “ “ “ “hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasn™t put into multivariate analysisenhanced not only by pdl1 amplification but also byjak2 activation [ ] therefore dlbcl with jak2pdl2 amplification was confirmed as an unique subtype that is different from dlbcl with myd88 l265pand othersobjective response rates orr of pd1 blockade therapy was “ in unselected patients with relapsedrefractory dlbcl [ ] the wide spectrum of orrmay be due to high heterogeneity of this subgroupansell sm study demonstrated patients with9p241 alteration in relapsedrefractory dlbcl inour cohort the frequency of jak2 and pdl2 amplification in relapsedrefractory dlbcl were and which were within the range of orr in the prior studies[ ] while patients were found thathad myd88 l265p mutation who may not be suitablefor antipd1 therapy thus the genetic analysis in refractoryrelapsed dlbcl is required for future therapyselection to increase the orr of immune checkpointinhibitorsjak2 amplification could augment the expression of itself and pd1 ligands pdl1 and pdl2 enhancing the 0cxue bmc cancer page of sensitivity to jak2 kinase inhibitor chemical jak2inhibition could reduce the rna transcription and protein expression of pdl1 thus selective inhibitionof jak2 would be a valuable complementary therapy forpdl1 blockadeauthors™ contributionsxx contributed to pdl1 ihc staining clinical followup data analysis andmanuscript writing wh contributed to ffpe tissues collection mlpa detection and clinical followup tq and lg provided experiment guidance anddata interpretation jy and nl contributed to study design coordination discussion and manuscript editing all authors read and approved the finalmanuscriptsjak2pdl2 exhibitsdlbcl with amplification ofpmbcllike cnas pattern and demonstrates unfavorable outcome resembling those with myd88l265p mutation thusit is essential to identify thissubgroup of dlbcl who may acquire more benefitsfrom the jak2 and pdl1 signaling inhibition andjak2 amplification detection by mlpa would be feasible in routine practice meanwhile the difference ofsurvival outcome between our study and wang j study indicated that pmbcllike dlbcl suggested by 9p241 cna could be an intermixed subgroup which required further explorationsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072933additional file mlpa results and clinical followup data the clinicopathological characteristics clinical followup data and mlpa results areshowed in this fileadditional file figure s1 representative results of mlparepresentative results of mlpa are showed in this figureadditional file table s1 the details of mlpa probes of genes indlbcl the locations and lengths of mlpa probes of genes are showedin this tableadditional file the detailed information of dlbcl with jak2pdl2amplification the detailed data about clinicopathological characteristicsmorphology immunohistochemistry and treatments of dlbcl with jak2pdl2 amplification are showed in this fileadditional file figure s2 the os and pfs of dlbcl with or withoutjak2pdl2_amp the os and pfs of dlbcl with or without jak2pdl2_ampadditional file figure s3 comparison of cnabased pattern andtheir survival outcome among pmbcl and three subgroups of dlbclone case of dlbcl with jak2pdl2 amplification and myd88 l265p mutation were included in dlbcl_myd88_l265p group a comparison ofcnabased patterns of driver genes among pmbcl and three subgroupsof dlbcl according to the status of jak2pdl2 amplification and myd88l265p mutation b survival curves and coxregression analysis of os andpfs among three subgroups of dlbcl after rchoplike treatmentadditional file figure s4 the frequencies of jak2 gain andamplification and their survival analysis a the frequencies of jak2 gainand amplification in dlbcl_jak2pdl2_amp and pmbcl b the os andpfs of dlbcl with jak2 gain or with jak2 amplificationabbreviationsdlbcl diffuse large bcell lymphoma pmbcl primary mediastinal large bcell lymphoma mlpa multiplex ligationdependent probe amplificationtcga the cancer genome atlas ipi international prognostic indexffpe formalinfixed paraffinembedded os overall survival pfs progressfree survival hr hazard ratioacknowledgementsnot applicablefundingthis study was partly supported by the beijing municipal science technology commission grant number z151100004015121 the cancerfoundation of china grant number lc2014l13 and cams innovation fundfor medical sciences grant number 2016i2m1001 to perform ffpe tissuescollection and mlpa detection and was partly supported by the cancerfoundation of china grant number lc2018b10 and pumc youth fundand the fundamental research funds for the central universities grantnumber to conduct pdl1 ihc staining and clinical followupand collect fish data of cmycavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its supplementary information filesethics approval and consent to participatethis is a retrospective study that was launched in november the casesenrolled in this project were diagnosed between jan and oct whose ffpe samples were used the data regarding treatment andprognosis were acquired by means of medical record consultation andtelephone conversation thus the need for consent was waived by theindependent ethics committee of cancer hospital chinese academy ofmedical sciences national gcp center for anticancer drugs ncc2015st05consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing china 2department ofpathology national cancer centernational clinical research center forcancercancer hospital shenzhen hospital chinese academy of medicalsciences and peking union medical college shenzhen chinareceived march accepted august referencesschmitz r wright gw huang dw johnson ca phelan jd wang jqroulland s kasbekar m young rm shaffer al genetics andpathogenesis of diffuse large bcell lymphoma n engl j med “chapuy b stewart c dunford aj kim j kamburov a redd ra lawrencems roemer mgm li aj ziepert m molecular subtypes of diffuse largeb cell lymphoma are associated with distinct pathogenic mechanisms andoutcomes nat med “godfrey j tumuluru s bao r leukam m venkataraman g phillip jfitzpatrick c mcelherne j macnabb bw orlowski r pdl1 genealterations identify a subset of diffuse large bcell lymphoma harboring a tcellinflamed phenotype blood “green mr monti s rodig sj juszczynski p currie t o'donnell e chapuy btakeyama k neuberg d golub tr integrative analysis reveals selective9p241 amplification increased pd1 ligand expression and furtherinduction via jak2 in nodular sclerosing hodgkin lymphoma and primarymediastinal large bcell lymphoma blood “ wang y wenzl k manske mk asmann yw sarangi v greipp pt krull jehartert k he r feldman al amplification of 9p241 in diffuse large bcell lymphoma identifies a unique subset of cases that resemble primarymediastinal large bcell lymphoma blood cancer j 0cxue bmc cancer page of lenz g wright gw emre nc kohlhammer h dave ss davis re carty slam lt shaffer al xiao w molecular subtypes of diffuse large bcelllymphoma arise by distinct genetic pathways proc natl acad sci u s a“swerdlow sh campo e harris nl jaffe es pileri sa stein h thiele j whoclassification of tumours of haematopoietic and lymphoid tissues revised4th edn lyon iarc cerami e gao j dogrusoz u gross be sumer so aksoy ba jacobsen abyrne cj heuer ml larsson e the cbio cancer genomics portal anopen platform for exploring multidimensional cancer genomics datacancer discov “gao j aksoy ba dogrusoz u dresdner g gross b sumer so sun yjacobsen a sinha r larsson e integrative analysis of complex cancergenomics and clinical profiles using the cbioportal sci signal pl1 nogai h wenzel ss hailfinger s grau m kaergel e seitz v wollertwulf bpfeifer m wolf a frick m ikappabzeta controls the constitutive nfkappab target gene network and survival of abc dlbcl blood “shi m roemer mg chapuy b liao x sun h pinkus gs shipp ma freemangj rodig sj expression of programmed cell death ligand pdl2 is adistinguishing feature of primary mediastinal thymic large bcelllymphoma and associated with pdcd1lg2 copy gain am j surg pathol“ qiu l zheng h zhao x the prognostic and clinicopathological significanceof pdl1 expression in patients with diffuse large bcell lymphoma a metaanalysis bmc cancer moelans cb monsuur hn de pinth jh radersma rd de weger ra vandiest pj esr1 amplification is rare in breast cancer and is associated withhigh grade and high proliferation a multiplex ligationdependent probeamplification study anal cell pathol amst “fernandezrodriguez c bellosillo b garciagarcia m sanchezgonzalez bgimeno e vela mc serrano s besses c salar a myd88 l265p mutation isan independent prognostic factor for outcome in patients with diffuse largebcell lymphoma leukemia “ ngo vn young rm schmitz r jhavar s xiao w lim kh kohlhammer h xuw yang y zhao h oncogenically active myd88 mutations in humanlymphoma nature “ dubois s viailly pj bohers e bertrand p ruminy p marchand vmaingonnat c mareschal s picquenot jm penther d biological andclinical relevance of associated genomic alterations in myd88 l265p andnonl265pmutated diffuse large bcell lymphoma analysis of casesclin cancer res “ gupta s cheville jc jungbluth aa zhang y zhang l chen yb tickoo skfine sw gopalan a alahmadie ha jak2pdl1pdl2 9p241amplifications in renal cell carcinomas with sarcomatoid transformationimplications for clinical management mod pathol “ ansell sm minnema mc johnson p timmerman jm armand p shipp marodig sj ligon ah roemer mgm reddy n nivolumab for relapsedrefractory diffuse large bcell lymphoma in patients ineligible for or havingfailed autologous transplantation a singlearm phase ii study j clin oncol“lesokhin am ansell sm armand p scott ec halwani a gutierrez mmillenson mm cohen ad schuster sj lebovic d nivolumab inpatients with relapsed or refractory hematologic malignancy preliminaryresults of a phase ib study j clin oncol “ hao y chapuy b monti s sun hh rodig sj shipp ma selective jak2inhibition specifically decreases hodgkin lymphoma and mediastinal largebcell lymphoma growth in vitro and in vivo clin cancer res “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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range of diseases including malignancies and autoimmune disordersIts high eï¬ectivenessprice ratio also won extensive application in ophthalmology On the other hand although MTX has anexcellent pharmacological efficacy MTX associated side eï¬ects in clinical use which vary from patient to patient are nonnegligible ere is no comparatively systematic review on MTX associated side eï¬ects and its risk factors is review aimed toreveal novel clinical approaches of MTX and its adverse eï¬ects in order to provide a reference for ophthalmic scholars in clinicalapplication of MTX IntroductionMethotrexate MTX is an antifolate metabolite that inhibitsDNA synthesis repair and cellular replication It was firstlyused as one of the essential treatments of pediatric leukemia[ ] According to previous studies MTX has also beenused to treat rheumatoid arthritis RA and psoriasis as anti‚ammatory and immunomodulatory agent [] as MTXcould not only optimize the efficacy of biological diseasemodifying antirheumatic drugs DMARDs [ ] but alsomake the therapeutic goals via lower doses in comparisonwith other conventional synthetic DMARDs [] Figure shows the pathway of folate in DNA synthesis the cellularpathway of MTX and how MTX works inside the cell Whileimmediate and lowdose MTX is used to treat nonmalignantand immunemediated disorders highdose MTX HDMTX more than mgm2week is widely used to treatmalignancies Until now HDMTX with or without radiation therapy is still the backbone of most modern chemotherapy regimens [] as well as the prevention ofsystemiccentral nervous system CNS lymphoma recurrence at a dose of gm2 per week []MTX has also been widely applied in ophthalmic diseases systemically and locally Recently published spay more attention to new clinical applications routes ofadministration and newly discovered side eï¬ects which arefoci of this review Clinical Applications in OphthalmologyAs one of the known corticosteroidssparing agents MTXhas been widely used in the treatment of anterior intermediate posterior or pan uveitis scleritis and ocularmucous membrane pemphigoid [] as well as advancedproliferative diabetic retinopathy [] However followedresearches reveal that MTX works with a significant difference in eï¬ectiveness ratings by anatomic location of‚ammation [] with treatment success achieved mostcommonly in patients with anterior uveitis and scleritis []In the treatment of noninfectious intraocular ‚ammationoral and intravenous are the most common routes with ausual dose range of mg to mg weekly e typical doseobserved was mgweek [ ] which is in the range oflowdose MTX e median time to achieve the success of 0cJournal of OphthalmologyFigure e cellular pathway of folate and MTX Dietary folate enters the cells through RFC1 as well as MTX In lowdose MTX treatmentMTX inhibits enzymes of the folate pathway Ultimately MTX leads to an increase in intracellular adenosine level which would cause anti‚ammatory eï¬ects RFC1 � reduced folate carrier ABC family � adenosine triphosphatebinding cassette ABC family DHF � dihydrofolate THF � tetrahydrofolate GGH � cglutamyl hydrolase FPG � folylpolyglutamate synthase MTXPG � methotrexate polyglutamate DHFR � dihydrofolate reductase MTHFR � methylene tetrahydrofolate reductase AICAR � aminoimidazole carboxamideribonucleotide ATIC � AICAR transformylasetreatment defined as control of ‚ammation with theability to taper corticosteroids to mg or less daily rangesfrom months to months for MTX [ ]Intravitreal MTX injection with or without systemicchemotherapy and radiotherapy has already been used totreat primary intraocular lymphoma patients [ “]According to Larkin [] intravitreal MTX injectioncould achieve remission in a proportion of patients withprimary intraocular lymphoma What is particularly noteworthy is that although MTX has a slow rate of onset ofeï¬ect when it was used to treat intraocular lymphoma viaintravitreal injection it prolonged local remission of oculardisease even with an aggressively growing tumor []erefore it has been taken as a relatively firstline choice forthe treatment of recurrent intraocular lymphoma [] although the treatment for primary intraocular lymphoma islacking solid justification because of the limited retrospectiveand prospective case series [] Local treatment via intraocular injection provides a consistent therapeutic MTXconcentration to reduce the systemic MTX associated sideeï¬ects [] erefore intraocular MTX injection is worthtrying especially for unilateral ocular diseases New Approaches of Applications of MTX MTX Used against Epithelial Downgrowth Previousstudies have already demonstrated safety of intravitrealMTX [] It has been used to treat intraocular lymphomaand proliferative vitreoretinopathy because ofits antiproliferative properties [] ere is a novel use of intravitreal MTX for recurrent epithelial downgrowth which wasnot treated by surgical and medical methods Lambert et al[] administered intravitreal MTX to patients with refractory proliferative membrane after cataract surgery whilemembrane peel and endolaser treatment failed e injectionof MTX was administered alone based on previous protocols and the presumed halflife of drugs in vitreous cavityAfter injections totally there was no membrane recurrence is case suggests that intravitreal MTX plays a role intreatment against epithelial downgrowth MTX Used in Conventional erapyResistant DiseasesGenerally the antivascular endothelial growth factor antiVEGF therapy has dramatically improved the prognosis ofneovascular agerelated macular degeneration nAMDHowever there are still some patients who remain refractoryto antiVEGF therapy which is termed as treatmentresistant nAMD As there is evidence that MTX has eï¬ects ininterrupting the angiogenesis cascade at various levels []Kurup oï¬ered intravitreal MTX to patients who wererefractive to standard antiVEGF therapy [] Although itwas an oï¬label use the patients™ visual acuity improved atfollowup visit while ophthalmic imaging examinationsshowed significantly reduced cystoid macular edema usFolatefolic acidRFC 1Folatefolic acidDHFTHFDHFR5MTHFMTHFRPurine and pyridine synthesis DNA synthesisMTXMTXABC familyFPGGGHMTXFGAICAR5FormylAICARATICAdenosine †‘AdenosineCell membraneCell membrane“““ 0cJournal of Ophthalmologypatients who are refractory to traditional antiVEGF therapymight benefit from intravitreal injection of MTXis approach is not alone Khalil [] had μg01 ml of MTX intravitreal injection once monthlyadministrated to adult Behcet™s disease BD patientssuï¬ering from BDassociated ocular ‚ammation withposterior segment involvement eir results prove thatintravitreal MTX improves visual acuity reduces posteriorsegment manifestations associated with Behcet™s disease andallows the reduction of corticosteroids and immunosuppressive drugs [] ese results also supported Taylor andassociates who conducted trials on patients with unilateral uveitis andor cystoid macular edema [] eirclinical trials suggest that intravitreal MTX may help patients with uveitisassociated posterior segment involvementto regain normal anatomical structure and then allowed thereduction of immunosuppressive therapy The Pharmacogenetics of MTXWith molecular sequencing and highthroughput technology large numbers of genetic polymorphisms can now bedetected accurately and rapidly [] Researchers pay moreattention to pharmacogenetics the study of genetic polymorphisms in drugmetabolizing enzymes and the translation of inherited diï¬erences to diï¬erences in drug eï¬ects[] e genes encoding transporting proteins and metabolizing enzymes for MTX are also known to harborfunctionally significant SNPs e SNPs may ‚uence theefficacy of MTX and have been suggested as potential riskfactors for enhanced MTX toxicity even in lowdose regimens based on previous researches []e research of pharmacogenetics of MTX could bedivided into genetic polymorphisms aï¬ecting MTX transport and SNPs that‚uence enzymes in the cellularpathway of MTX []Once taken MTX enters the cell through an activetransport which is mediated by the reduced folate carrier RFC1 e loss of RFC1 gene expression might lead toeï¬ects of uptake and intracellular levels of MTX A G80ASNP of RFC1 was proposed [] making a decreasing [] orincreasing [ ] eï¬ect on intracellular level of MTXerefore a significant association between RFC1 SNPs andMTX toxicity should be considered Chango state thatthese SNPs strongly impact the overall MTX associated sideeï¬ects by resulting in altered cellular MTX concentrationbut with no ‚uence on MTX efficacy [] However someresearchers argue that these SNPs have no definite eï¬ect[] us it remains controversial whether SNPs of RFC1aï¬ect the transport of MTX Moreover Pglycoprotein amembrane transporter that has ‚uences on the dispositionand bioavailability of MTX [] was studied SNPs ofABCB1 including C3435T SNP and C1236T SNP werebelieved to have eï¬ects on the expression of Pglycoprotein[] Gervasini speculate the C1236T SNP of ABCB1aï¬ects the administered doses of MTX and the incidence ofhematological toxicity [] However just like G80A SNPthere are disputes about the ‚uences of these SNPs asdiï¬erent studies had diï¬erent outcomes []Metabolizing enzymes were also being analyzed giventhe critical role of transporters in disposition of MTX and itsactive products as well as the folate metabolism MTXpharmacogenetics mostly focused on the SNPs in theMTHFR gene e present study shows that genetic polymorphisms in the folate metabolic pathway and in MTXtransporters ‚uence the toxicity but not the efficacy of thelowdose MTX treatment in patients with autoimmunediseases [] For example C677T and A1298C are knownin MTHFR gene to result in a lower enzyme activity []Windsor and associates reported that MTHFR A1298C andC677T were associated with MTX related nephrotoxicityand anemia [] ese SNPs might be associated withdecreased activity of methylenetetrahydrofolate reductaseelevated plasma homocysteine levels and altered distribution of folate [] us patients with this genotype weremore vulnerable to potential MTX induced toxicity sincethese reactions above may lead to slower folate metabolismand slower cell repair [] Weisman used univariatelogistic regression to reveal that the MTHFR C677T alsoincreases the occurrence of side eï¬ects in central nervoussystem manifested as headache and lethargy [] HoweverLambrecht argued that MTHFR C667T was not apredictive factor for toxicity [] Berkani found noassociation between A1298C polymorphism and MTXtoxicity [] Interestingly Grabar claimed that thepatients with MTHFR 1298C genotype have a lower risk forMTX toxicity than the carriers of MTHFR 1298A allele []To date the study of pharmacogenetics of MTX continues An increasing number of SNPs have been found to bepossibly associated with the efficacy and toxicity of MTXe newly discovered genotypes include C347G in ATICand ²UTR 28bp repeat and ²UTR 6bp deletion inTYMS which may ‚uence both efficacy and toxicity ofMTX similarly factors that may aï¬ect MTX associatedtoxicity are for example A2756G in MS and A66G in MTRR[ ] e genes and their SNPs that might beassociated with the eï¬ects and side eï¬ects of MTX aresummarized in Table Growing evidences suggest that asingle genetic factor is unlikely to adequately predict theefficacy and toxicity of MTX in polygenic disease such as RAand autoimmune associated ocular disease Given the impactof MTX in several metabolic pathways a complex of multiple risk genotypes examination would help to predict theefficacy of MTX and to identify patients who may haveadverse eï¬ects from MTX administrationTaken together the efficacy and toxicity of MTX mayremain associated with the genetic markers in the patientserefore although this remains a controversial subject it isreasonable to believe that pharmacogenetics may be able topredict who is at risk of MTX associated adverse eï¬ects andmay help in maximizing the benefitrisk ratio of MTX The Side Effects of MTXe doselimiting toxicity of MTX mainly includes hepatotoxicity and nephrotoxicity [“] but mortality hasoften been reported due to either pneumonitis or secondaryinfections [] 0cJournal of OphthalmologyTable Summary of genes and their SNPs which might have possible clinical eï¬ects towards MTXTransportingproteinsABCfamilyGeneRFC1 [“]ABCB1 [ ]ABCC1 []ABCC2 []ABCC4 []MetabolizingenzymesMTHFR [“]ATIC [“]TYMS [ ]GGH [ ]DHFR [ ]MS [ ]MTRR [ ]SNPsG80AC3435TC1236Trs246240Srs3784862A2412GG1249AG1058AC934AC677TA1298CC347G²UTR 28bp²UTR 6bprepeatdeletionC452TC401TT721AC830TA2756GA66GSome experts divided MTX associated pulmonarycomplications into ‚ammatory infectious and lymphoproliferative [] In the authors™ opinion all MTX relatedside eï¬ects can be classified into these three categoriesaccording to the pharmacological eï¬ects of MTXMajor adverse events for MTX are related to the folateantagonism and primarily aï¬ect highly proliferative tissuessuch as bone marrow and gastrointestinal mucosa []Given the immunosuppression eï¬ect of MTX pancyt iawas one of the most frequent severe toxicities of methotrexate [] Meanwhile the risk of developing an infectiousprocess is increased all along the treatment and the severityof the infected disease would be worsen [ ] includingcommon bacterial infections herpes zoster eruptions andopportunistic infections According to previous studies therisk is larger than that with other disease modifying antirheumatic nonbiological drugs DMARDsSecondly the MTX acts as the hapten [] and is likely toreact directly with nucleophilic groups present in proteins ieto combine with endogenous protein [ ] e proteinadducts thus act as an antigenic signal to direct the eï¬ector armof the immune response [] e provoked immune responsesare most commonly type I immediate hypersensitivity andtype III immune complex reactions [] Hypersensitivitypneumonitis is the most common severe and unpredictablecomplication with a mortality of up to almost []Moreover a few studies have shown that longterm MTXuse can lead to lymphoproliferative disorders LPDs in bothnodal sites and extra nodal sites such as the skin lungsepipharynx thyroid gland nasal cavity spleen and kidneysespecially for patients who are positive for EBV infection[“] e reported frequency of EBV positive in MTXassociated LPDs patients is “ [] Although themechanism of onset is not fully understood it is believedPossible clinical eï¬ectsIncreasingdecreasing intracellular MTX levelAï¬ecting efficacy of MTXAï¬ecting the distribution of MTX and incidence of hematological toxicityAssociation with MTX related toxicityLeading to accumulation of MTX to nephrotoxic levelsAssociation with MTX related gastrointestinal toxicityAssociation with MTX related hepatotoxicityAssociation with MTX related hematological toxicityAï¬ecting the toxicity but not the efficacy by resulting in a lower enzymeactivity association with related nephrotoxicity anemia and neurologicside eï¬ectsAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAssociation with MTX associated toxicityAssociation with MTX associated toxicitythat the combination of immunodeficiency and the immunosuppressive eï¬ect of MTX has been implicated in thepathogenesis of MTX associated LPDs e World HealthOrganization WHO has classified MTX associated LPDs aslymphoid neoplasms whether iatrogenic or immunodeficiency associated diseases [ ] MTX associated LPDsoften take a spontaneous remission which tends to completemostly within weeks after the discontinuation of MTX[] But there are a few reports showing that the lymphoidneoplasms occur even after stopping using MTX [] e Eï¬ects of Administration Routes Generally the sideeï¬ects of MTX depend on the route of administration Dosedependent [] gastrointestinal side eï¬ects are the mostfrequent side events with orally administered MTX as oraladministration is the most common delivery method[ ] More than of MTX is excreted by therenal system thus MTX associated nephrotoxicity is common among patients taking MTX Fortunately the resolution usually occurs after discontinuation of therapy andsalvage treatment with highdose corticosteroids[]erefore to achieve treatment with less side eï¬ects theappropriate route of administration and dose of MTX arenecessary During the treatment monitoring of patients™general condition mattersAdverse eï¬ects of intravitreal injections of MTX occuronly within the eyeincluding hyperemia keratopathycataract iridocyclitis vitreous hemorrhage retinal detachment maculopathy and endophthalmitis []Splitting doses of MTX rather than intravenous administration is a new attempt to avoid MTX associated sideeï¬ects MTX is split and given twice or thrice in a week toachieve higher bioavailability and better clinical response 0cJournal of Ophthalmology[ ] thus providing us with a novel method of oraladministration of MTX with less adverse eï¬ects Is LowDose MTX Safer Based on clinical cases observation side eï¬ects which can lead to discontinuation ofMTX are rare during the typical ophthalmology treatmentbecause of the lower dose of MTX required [] e application of lowdose MTX regimen has also become one ofthe main therapies of a variety of immunemediated diseasesbecause of its efficacy and an acceptable safety profile asmost lowdose MTX associated toxicity has been describedin case reports and relatively small case series []However although welltolerated and mostly reversibleeven a lowdose regimen of MTX can result in clinicallysignificant toxicity with substantial death rates about according to Kivity™s cohort study [] e lowdose MTXassociated severe adverse eï¬ects include major centralnervous system complications [] mucositis pulmonaryinvolvement hepatotoxicity [] and myelosuppression Is MTX Safe to the Pregnant and Fetuses As one of thelipidsoluble and low molecular weight drugs MTX could bereadily transferred across the placental membrane duringpregnancy and adversely aï¬ect the fetus [] In addition MTXmight take longer time for elimination in fetal tissues []Regarding pharmacogenetics mutations caused by MTXlead to severe decrease of the expression of folate and nucleobaseenzymes which are critical for cellular homeostasis [] Inpractice MTX aï¬ected formation of the blastocyst and causeddysmorphic features and neurologic defects in early pregnancyleading to malformations in some cases [] Multiple congenitalabnormalities have been observed after weekly MTX treatmentsat a mg dose during the first months of pregnancy [] evenfetal death [] Verberne had reviewed cases of congenitalanomalies after in utero exposure to MTX and proved that somecongenital anomalies such as microcephaly craniosynostosistetralogy of Fallot pulmonary valve atresia limb reductiondefects and syndactyly were truly part of the œfetal methotrexatesyndrome [] Administration of MTX in childhood mightalso cause manifestations including visual defect [] andSmith“Magenis syndrome [] among patients with specificmutations us special care should be taken with pregnantpatients and children in particular e Risk Factors of MTX Associated Side Eï¬ects e mostcommon risk facts of MTX induced adverse eï¬ects areadvanced age age years and underlying disease incaused by MTX administration with100 g alcohol concluding renal andor hepatic insufficiency and lung diseaseespecially patients with chronic hepatitis B and diabetesmellitus [“] Patients with a history of alcohol intakemight have a greater risk of liver fibrosis and hepatotoxicitysumption per week [] Also preexisting hypoalbuminemiaand past use of any of the DMARDs and protonpumpinhibitors have been described in studies to increase theincidence of MTX induced side eï¬ects [ ] Moreovertaking drugs that may interact with MTX at the same timemight also be dangerous these drugs include salicylatescotrimoxazole chloramphenicol sulfonamides cyclosporine and pyrimethamine [] Although no significantprotective eï¬ect of folate supplementation on MTX relatedtoxicity has been found [] the folate deficiency is anotherreason for the side eï¬ects based on clinical cases []Heidari found that MTX administration elevatedkidney ROS levels decreased tissue antioxidant capacityincreased lipid peroxidation and depleted renal glutathionestores eir research data indicate that MTX caused tissuedamage and organ dysfunction through oxidative stresserefore they proposed that patients with preexistingmitochondrial defects might be vulnerable to MTX inducedrenal injury []e use of highdose MTX HDMTX is also the riskfactor of adverse eï¬ects MTX induced liver fibrosis is morelikely to become morphologically evident with high cumulative doses possibly largely exceeding to mg[ ] and the side eï¬ects caused by omeprazole use inthe past were found in cancer patients receiving HDMTXtreatment []e distribution of MTX in vivo also plays a role in MTXrelated side eï¬ects As MTX tends to accumulate in theextravascular compartment patients with pleural eï¬usionascites and massive edema should get extra caution due tothe risk of toxicity from reabsorption of extravascular fluid[]Another noteworthy risk factor is UV UV recall phenomenon also known as MTX associated UV reactivationhas been reported [ ] It is reactivity of sunburn areaswithin to days of the treatment with MTX [ ]According to Adams and associates this phenomenon mightbe due to the immune response by uncontrolled sunburninduced ‚ammation released by MTX [] Patients whopreviously suï¬ered sunburns deserve more detailed monitoring when methotrexate is needed Is Folate Supplementation Necessary for OphthalmicPatients To prevent MTX associated side eï¬ectsit iscommon to take folate [as either folic acid FA or folinicacid FLA] in clinic [ ] However there is noconsistent and evidencebased guideline for folate supplementation in ophthalmic patientsFolate and folic acid play significant roles in the de novosynthesis of purines and thymidylate which are required forDNA replication and repair [] Funk and associates founda significant reduction of circulating folate concentrations in of patients receiving MTX treatment [] Patientstreated with highdose MTX HDMTX got routine folatesupplementation to reduce HDMTX associated side eï¬ects[“] After a systematic literature review of HDMTXtherapy and folate supplementation Van der Beek et al[] found lower incidence of MTX associated adverseeï¬ects in regimens with higher cumulative doses and earlieradministration of folate supplementation in similar HDMTX dosage studies Folate supplementation in patientswith lowdose methotrexate is also being studied Ortiz et al[] had proved the protectivefolateeï¬ectof 0cJournal of Ophthalmologysupplementation by conducting a Cochrane review including more than patients taking lowdose MTX Untilnow folate supplementation had been proved to prevent andimprove MTX associated eï¬ects including gastrointestinalrespiratory and neurologic side eï¬ects [ ] Mori et alsupported the protective eï¬ect by demonstrating that patients treated with lowdose MTX without folate supplements were significantly associated with the development ofmyelosuppression and pancyt ia []However Arabelovic and associates™ preliminary studyshowed a significant increase of MTX dose needed []since folic acid fortification enriched cereal grain productswere fully implemented in the USA and Canada [] isconveyed a message to us that high dose of folate supplementation might have ‚uence on the efficacy of MTXAlDabagh found that the reduction in efficacy ofMTX cannot be ignored while folate supplementation didmake a significant reduction in associated adverse eï¬ects[] Salim declared the decreasing ‚uence betweenthe anti‚ammatory eï¬ect of MTX and folate supplementation by carrying out a doubleblind clinical trial []Chladek had conducted an labeltwowaycrossover study supporting the opinion above [] Additionally because of the unequal distribution of folic acidand MTX in organs and tissues [] MTX discontinuationis more common for some MTX associated side eï¬ects inophthalmic clinic [] rather than higher dosage of folatesupplementationere are no ophthalmic studies to demonstrate theprotective eï¬ects of folic acid supplementation us although the folate supplementation is widely used amongpatients treated with lowdose MTX [ ] the necessityand standardized dosage of folate supplementation in specific patients [] as well as the MTXfolate interactionstill warrant further studies DiscussionMethotrexate as one of the alternative pharmacologicalsteroidsparing immunosuppressive agentsis becomingmore and more popular as the preferred treatment in severalautoimmune conditions requiring longterm immunosuppression [] Lowdose MTX has anti‚ammatory andimmunomodulatory properties by increasing levels of intracellular and extracellular adenosine [] which is thefoundation of ophthalmic MTX treatment e standardizedand recommended administration of ophthalmic MTXtreatment is once a week starting with a dose of mg andescalating every to weeks up to “ mgweek whennecessary [ ] In patients with insufficient response toMTX alone cyclosporin with or without azathioprine wasadded []To avoid side eï¬ects split doses of MTX administrationand folate supplementation are gradually being used inophthalmic clinic Prescription of to mg of folatesupplementation has a significant role in MTX safety []but the higher dosage is less applied even with higher dose ofMTX [] Prophylactic folate supplementation is notnecessary in most patients [] ere is also research toconvey that ml100 g or above dosage of fish oil is aseï¬ective as folinic acid in therapeutic potential in preventingbone loss during MTX chemotherapy [] For some resistant andor mortal adverse eï¬ects the discontinuation ofMTX will work instantlyWith the increasing longterm use of MTX it is importantto monitor patients™ blood examination results including bloodroutine and liver and renal functions As pancyt ia can be alate manifestation [] elevation of urea creatinine aminotransferases and albumin as well as electrolytes disturbancesmay result in MTX associated liver and renal side eï¬ects []Plasma MTX level is not a reliable predictor for adverse eventsin MTX therapy [] On the contrary circulating folate levelsand folate polyglutamate distribution change sensitively withMTX exposure and exogenous folate supply [] and could beused as a biomarker of MTX efficacy [] It should be notedthat as erythrocytes have a halflife of approximately daysthe results of blood examinations might reflect both pretreatment and posttreatment status which need to be analyzedcarefully []Numerous studies had been conducted to prove thatMTX could be used as a welltolerated safe and eï¬ectivefirstline treatment Hence the MTX administration shouldnot continue to be stigmatized as a œcancer drug or to bediscouraged because of associated adverse eï¬ects Contrarily the indication and the routes of administration areabout to gradually widenConflicts of Intereste authors declare that they have no conflicts of interestReferences[] R Q H Kloos R Pieters C van den Bos œe eï¬ect ofasparaginase therapy on methotrexate toxicity and efficacy inchildren with acute lymphoblastic leukemia Leukemia Lymphoma vol no pp “ [] R K Bath N K Brar F A Forouhar and G Y Wu œAreview of methotrexateassociated hepatotoxicity Journal ofDigestive Diseases vol no pp “ [] W Wang H Zhou and L Liu œSide eï¬ects of methotrexatetherapy for rheumatoid arthritis a systematic review European Journal of Medicinal Chemistry vol pp “[] J Smolen and R Landew´e œEULAR recommendations forthe management of rheumatoid arthritis with synthetic andbiological diseasemodifying antirheumatic drugs update Annals of the Rheumatic Diseases vol no pp “ [] J A Singh K G Saag S L Bridges œ Americancollege of rheumatology guideline for the treatment ofrheumatoid arthritis Arthritis Rheumatology vol no pp “ [] M Holdhoï¬ P Ambady A Abdelaziz œHighdosemethotrexate with or without rituximab in newly diagnosedprimary CNS lymphoma Neurology vol no pp “ [] J Pe™er J M Rowe S Frenkel and E J Dann œTesticularlymphoma intraocularvitreoretinal lymphoma and brainlymphoma involvement of three immunoprivileged sites in 0cJournal of Ophthalmologyone patient American Journal of Hematology vol no pp “ [] S Gangaputra C W Newcomb T L Liesegang et alœSystemic immunosuppressive therapy for eye diseases cohort study methotrexate for ocular ‚ammatory diseasesOphthalmology vol no pp “ [] P W Hardwig J S Pulido J C Erie K H Baratz andH Buettner œIntraocular methotrexate in ocular diseasesother than primary central nervous system lymphomaAmerican Journal of Ophthalmology vol no pp “ [] E Esterberg and N R Acharya œCorticosteroidsparingtherapy practice patterns among uveitis specialists Journalof Ophthalmic Inflammation and Infection vol no pp “ [] K Durrani F R Zakka M Ahmed M MemonS S Siddique and C S Foster œSystemic therapy withconventional and novel immunomodulatory agents for ocular ‚ammatory disease Survey of Ophthalmology vol no pp “ [] S S Gangaputra C W Newcomb M M Joï¬e et alœComparison between methotrexate and mycophenolatemofetil monotherapy for the control of noninfectious ocular‚ammatory diseases American Journal of Ophthalmologyvol pp “ [] V K Ayuso E L van de Winkel A Rothova and J Helenade Boer œRelapse rate of uveitis postmethotrexate treatmentin juvenile idiopathic arthritis American Journal of Ophthalmology vol no pp “ [] S R Rathinam M Babu R undikandy œA randomized clinical trial comparing methotrexate and mycophenolate mofetil for noninfectious uveitis Ophthalmologyvol no pp “ [] A Galor D A Jabs H A Leder œComparison ofantimetabolite drugs as corticosteroidsparing therapy for‚ammation Ophthalmologynoninfectiousvol no pp “ ocular[] M D de Smet V S Vancs D Kohler D Solomon andC C Chan œIntravitreal chemotherapy for the treatment ofrecurrent intraocular lymphoma British Journal of Ophthalmology vol no pp “ [] E Kim C Kim J Lee and Y Cho œA case of primaryintraocular lymphoma treated by intravitreal methotrexateKorean Journal of Ophthalmology vol no pp “[] J Smith J T Rosenbaum D J Wilson œRole ofintravitreal methotrexate in the management of primarycentral nervous system lymphoma with ocular involvementhistorical image Ophthalmology vol no pp “ [] CC Chan and D J Wallace œIntraocular lymphomaupdate on diagnosis and management Cancer Controlvol no pp “ [] K L Larkin U S Saboo G M Comer œUse ofintravitreal rituximab for treatment of vitreoretinallymphoma British Journal of Ophthalmology vol no pp “ [] C P Fox E H Phillips J Smith œGuidelines for thediagnosis and management of primary central nervoussystem diï¬use large Bcell lymphoma British Journal ofHaematology vol no pp “ [] A Sadaka R Sisk J Osher O Toygar M Duncan andinfusion forœIntravitreal methotrexateC Riemannproliferative vitreoretinopathy Clinical Ophthalmologyvol pp “ [] N G Lambert DJ Wilson D M Albert andW D Chamberlain œIntravitreal methotrexate for recurrentepithelial downgrowth JAMA Ophthalmology vol no p [] A M Joussen F E Kruse HE V¨olcker and B KirchhofœTopical application of methotrexate for inhibition of cornealangiogenesis Graefe™s Archive for Clinical and ExperimentalOphthalmology vol no pp “ [] S K Kurup C Gee and C M Greven œIntravitrealmethotrexate in therapeutically resistant exudative agerelated macular degeneration Acta Ophthalmologica vol no pp e145“e146 [] H E M Khalil H A Raafat N A Azab H E Haroun andH A Elgendi œe role of intraocular methotrexate in themanagement of uveitis and posterior segment involvementin Behçet™s disease patients e Egyptian Rheumatologistvol no pp “ [] S R J Taylor A Banker A Schlaen œIntraocularmethotrexate can induce extended remission in some patients in noninfectious uveitis Re
2
" 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 ˚c 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 bio‘tek 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 bio‘tek 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 anti‘n‘cadherin antibody ab18203 dilution antiecadherin antibody ab1416 dilution anti‘mmp‘ ab38898 dilution anti‘active‘caspase‘ antibody ab2324 dilution antiactivecaspase3 antibody ab2302 dilution antiapaf1 antibody ab2324 dilution anti‘pi3k‘p85 antibody ab191606 dilution antipi3k110 antibody ab32569 dilution anti‘pan‘akt phospho t308 antibody ab38449 dilution anti‘pan‘akt antibody ab8805 dilution anti‘pdk1 antibody ab52893 dilution and anti‘gapdh ab9484 dilution the secondary antibody was selected as follows goat anti‘rabbit ab150077 dilution or goat anti‘rat ab150117 dilution the blocking protocol was with skim milk for h at room temperature the primary antibody was incubated overnight at ˚c 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 ˚c 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 bio‘tek 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 ˚c 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 pre‘cooled ethanol pre‘chilled pbs and water‘free configuration was added at ˚c 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 ‘Ëšc 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 rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 emt epithelial‘mesenchymal 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 inhi‘bition 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 rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 apaf1 apoptotic protease activating factor the expression of e‘cadherin 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 rg35‘fu group ‘fu ‘fluorouracil rg3 ginsenoside rg3 cdk cyclin‘dependent 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 p‘p85 p‘110 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 rg35‘fu 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 rg35‘fu 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 c‘e 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 rg35‘fu 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 g1s‘specific 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 and stage ‘ cancer epidemiol biomarkers prev ‘ siegel rl miller kd and jemal a cancer statistics ca cancer j clin ‘ chen w zheng r baade pd zhang s zeng h bray f jemal a yu xq and he j cancer statistics in china ca cancer j clin chen w zheng r zhang s zeng h zuo t xia c yang z and he j cancer incidence and mortality in china in an analysis based on urbanization level chin j cancer res ‘ li j hou n faried a tsutsumi s and kuwano h inhibition of autophagy augments ‘fluorouracil chemotherapy in human colon cancer in vitro and in vivo model eur j cancer sanoff hk carpenter wr freburger j li l chen k zullig ll goldberg rm schymura mj and schrag d comparison of adverse events during ‘fluorouracil versus ‘fluorouraciloxaliplatin adjuvant chemotherapy for stage iii colon cancer a population‘based analysis cancer ‘ cheah ky howarth gs bindon ka kennedy ja and bastian sep low molecular weight procyanidins from grape seeds enhance the impact of ‘fluorouracil chemotherapy on caco‘ human colon cancer cells plos 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 e331‘e333 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
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Integrinmediated adhesive properties ofneutrophils are reduced by hyperbaric oxygentherapy in patients with chronic nonhealingwoundMonica BaiulaID1 Roberto Greco2 Lucia Ferrazzano2 Alberto Caligiana1Klarida Hoxha3 Daniele Bandini3 Pasquale LongobardiID3 Santi Spampinato1˜¯Alessandra TolomelliID2˜¯ Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna Bologna Italy Department of Chemistry œGiacomo Ciamician Alma Mater Studiorum University of Bologna BolognaItaly Hyperbaric Centre Ravenna Italy˜¯ These authors contributed equally to this work Current address MediNeos Observational Research Modena Italy alessandratolomelliuniboit AT santispampinatouniboit SSa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Baiula M Greco R Ferrazzano L CaligianaA Hoxha K Bandini D Integrinmediated adhesive properties of neutrophils arereduced by hyperbaric oxygen therapy in patientswith chronic nonhealing wound e0237746 101371journalpone0237746Editor Nukhet AykinBurns University of Arkansasfor Medical Sciences College of Pharmacy UNITEDSTATESReceived May Accepted July Published August Copyright Baiula 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 All relevant data arewithin the manuscript and its SupportingInformation filesFunding Financial Disclosure This researchstudy was supported by Fondazione delMonte di Bologna e Ravenna wwwfondazionedelmonteit through a grant to ATFdM3980 prot N˚ bis2015 The authorswere also supported by the University of BolognaIn recent years several studies suggested that the ability of hyperbaric oxygen therapyHBOT to promote healing in patients with diabetic ulcers and chronic wounds is due to thereduction of inflammatory cytokines and to a significant decrease in neutrophils recruitmentto the damaged area α4 and 2 integrins are receptors mediating the neutrophil adhesionto the endothelium and the comprehension of the effects of hyperbaric oxygenation on theirexpression and functions in neutrophils could be of great importance for the design of noveltherapeutic protocols focused on antiinflammatory agents In this study the α4 and 2 integrins™ expression and functions have been evaluated in human primary neutrophils obtainedfrom patients with chronic nonhealing wounds and undergoing a prolonged HBOT kPaper minutes The effect of a peptidomimetic α41 integrin antagonist has been also analyzed under these conditions A statistically significant decrease in 2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one monthafter the last treatment while α4 integrin levels remained unchanged However cell adhesion function of both neutrophilic integrins α41 and 2 was significantly reduced and respectively but α41 integrin was still sensitive to antagonist inhibition in the presence of fibronectin suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacyIntroductionHyperbaric oxygen HBO therapy has emerged in the last years as an innovative approachand an effective adjunctive therapy for the treatment of different pathologies The oxygenPLOS ONE 101371journalpone0237746 August PLOS ONE 0cRFO17 RFO18 RFO19 and by Italian Ministryof Education University and Research with aspecific research project PRIN project20157WW5EH and a grant to the Department ofChemistry œGiacomo Ciamician of the Universityof Bologna under the initiative Department ofExcellence L232 del The fundershad no role in study design data collection andanalysis decision to publish or preparation of themanuscriptCompeting interests The authors have declaredthat no competing interests existIntegrins™ role in HBOTpromoted wound healingpressure applied in the chamber is usually from to kiloPascal kPa“ to absoluteatmospheres ATA and the first effect of pressurizing the human body is the increase of partialpressure of gases and the decrease of volume of gasfilled spaces according to Boyle™s law [ ]The additionally available oxygen has the ability to restore oxygenation in areas where hypoxia or hypoperfusion occur and it can help damaged tissue to heal [] Moreover increasedoxygen levels that lead to changes in reactive oxygen species ROS and nitrogen speciesRNS production during HBO therapy HBOT are essential to stimulate specific repair functions of macrophages neutrophils and fibroblasts in the healing process [“] In additionHBOT regulates the inflammatory response reduction of NLRP3 inflammasome proinflammatory cytokines including IL1 IL6 e IL18 TNFα [“]HBOT has been successfully employed to control nonhealing diabetic ulcers and chronicwounds significantly minimizing the number of amputations relative to standard wound carealone in diabetic population []Wound healing is a complex process that involves growth factors components of the extracellular matrix and several cell types Inflammatory cytokines such as tumor necrosis factorαTNFα and interleukin1 IL1 are often present at high levels in the site of inflammationlike in chronic wounds [] The immune system is involved in all the steps of tissue repair[] Inflammatory response evolves in the leukocyteadhesion cascade primarily mediated bytwo major adhesion receptor families selectins and integrins []Neutrophils play a crucial role in wound healing process by sensing their environment andresponding to the extracellular signals by adhesion migration and other effector functions[] After ending their role at the site of inflammation neutrophils undergo apoptosis and areremoved by macrophages this latter event is considered a strong signal for inflammation resolution Although fighting infection neutrophils can also have harmful effects inducing damagein the inflamed tissue and leading to a delay in healing process [] and chronic inflammationNeutrophils express integrins on their surface significantly contributing to the recruitmentphase Among them 2 integrin family members including αL2 and αM2 bind to endothelial intercellular adhesion molecule1 ICAM1 and α41 integrin recognizes vascular celladhesion molecule1 VCAM1 expressed on endothelial cells [] Moreover α41 integrinand 2 integrins are involved in the onset and resolution of inflammatory process mediatingthe adhesion of monocytes lymphocytes and neutrophils to the blood vessels []Several studies carried out on animal models have evidenced that neutrophil recruitment issignificantly reduced by treatment with HBO in case of damage induced by ischemia andreperfusion [“] in other studies HBOT induces the reduction of tissue necrosis [ ]and lipid peroxidation []Moreover it has been shown that HBO treatment inhibits ischemia reperfusioninducedneutrophil adhesion to endothelium by blocking 2 integrin polarization [ ] and may alsoreduce leukocytes recruitment as it impaired adhesion molecule function by Snitrosation[] Conversely the role played by α41 integrin in neutrophilmediated adhesion to endothelium has been poorly investigated but an important role of α41 in 2 integrinindependentmigration of neutrophils across heart endothelium has been demonstrated in vitro suggestinga similar in vivo situation in neutrophil trafficking in reperfused myocardium []HBOT represents an effective therapy for chronic wounds as it reduces inflammation andaccelerates healing [] probably involving integrins In the present study we investigatedwhether HBOT could exert its effects by modulating functions of α41 and 2 integrinsexpressed on neutrophils obtained from patients with chronic nonhealing ulcers In a previous study Thom [] isolated polymorphonuclear leukocytes PMN from young healthyvolunteers exposed to only one session of HBO They observed a reduction in cell adhesionmediated by 2 integrins without any variation of its expression Our aim is to evaluate the rolePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingof integrinmediated adhesion by characterizing the expression of integrin receptors on neutrophils during the HBOT and by analyzing the effect of a peptidomimetic α41 integrin antagonist under these conditions If integrins are a target for both HBOT and synthetic antagonistthat blockade their activation a joint therapy could be hypothesized leading to a faster andstronger decrease of inflammation To this purpose expression and function of α41 and 2integrins were investigated for the first time in human primary neutrophils isolated from theblood of patients with chronic nonhealing ulcer undergoing HBOT or standard wound therapy alone Expression of these integrins was monitored in patients before the beginning ofHBOT exposure and during the therapy using specific antibodies towards α4 integrin or 2integrin family Patient wound area size was measured and proinflammatory cytokine levelswere evaluated both in neutrophils and in plasma Furthermore in vitro cell adhesion assayswere performed in the presence of a peptidomimetic integrin antagonist previously developedby our group [] to investigate if the HBO treatment may influence neutrophil recruitmentand adhesion mediated by α41 integrinMaterials and methodsPatients™ recruitmentThe study protocol was conformed to the ethical guidelines of the Declaration ofHelsinkiThe patients followed exclusively the therapy prescribed by their medical doctor followingthe indications of the Italian public health system No additional treatments were done for thepurposes of the study which did not change any therapeutic protocol nor interfere with thehealing progress The patients of the HBOT group were submitted to the prescribed HBOTand to blood sampling following the standard protocols approved by the Institutional EthicsReview Board of the Comitato Etico della Romagna CEROM Version of June approved by CEROM Ethical Committee Reg Sperimentazioni Prot N45332018I5150 which has full access to the data in order to check conformity of the study to thecurrent regulationThirty patients from Hyperbaric Centre Ravenna Italy were enrolled in the study betweenJuly and January in two different groups control group patients n receivedstandard wound care alone as prescribed by their medical doctor whereas HBOT grouppatients n received HBOT in addition to conventional wound treatment The inclusioncriteria were adults years or older and chronic wounds that fail to demonstrate improvement wound area reduction after a minimum of weeks of standard wound therapyThe exclusion criteria were symptoms of bacterial infection malignancy pregnancy medications that can adversely affect healing including anticonvulsants steroids antibiotics angiogenesis inhibitors and NSAIDs such as drugs known to promote healing including vitaminsthyroid hormone and iron Contraindication for HBOT were claustrophobia uncontrolleddiabetes middle ear problems glaucoma heart failure pacemaker untreated pneumothoraxchronic obstructive pulmonary disease pulmonary emphysema with retention of CO2 prothesis and seizures Patients in the control group were matched by gender age and ethnicityExclusion criteria for control group patients were the same for patients in HBOT groupThe patients allowed donating blood samples and were informed of the aim of the studyand completed the written informed consent process before enrolling in the study At the endof the study all individuals received personal information about the results relative to theirown samplesThe following demographic information was collected for all recruited patients at baselinegender age smokernonsmoker and other medical conditions Photographs of the ulcer werePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingtaken at various times After sharp wound debridement with a sterile scalpel the wound™s surface area was calculated by VISITRAK„¢ Digital System Smith Nephew For each patientthe ulcer was graded and staged by a clinician as described belowSample sizeSample size was based on a power analysis using G�Power [] The power was set at tolimit the risk of committing a type II error to the α level was set at The effect sizewas set at and the number of samples for each group was calculated to be to detect statistically significant differences P value of between groups In this study patientswere selected and allocated to study groups control and HBOT groupHyperbaric oxygen therapy protocolHBOT was conducted at the Hyperbaric Centre in Ravenna Italy The protocol procedureconsisted of HBO exposures in a multiplace hyperbaric chamber daily session five perweek from Monday to Friday HBOT group patients breathed oxygen at kPa per minutes in cycles for minutes separated by minutes medical air breathing intervalsClassification of woundsIn this study ulcers were graded using Falanga Wound Bed Preparation Score Staging copyrighted [] that provides staging of varying degrees based upon descriptions and characteristics of ulcers Table [] Staging of the wounds was done by combining the score of thewound bed appearance with that of the wound exudate Table Clinical assessment ofwound conditions was conducted for HBOT group before HBO treatment T0 immediatelyafter the fifteenth HBOT session T15 and one month after ending HBOT T1M for controlgroup during the first evaluation by a medical doctor T0 and after fifteen days of conventional wound therapy T15Neutrophil isolation from peripheral bloodVenous blood samples were obtained from the antecubital vein of participants in EDTAcontaining vacutainers Neutrophil isolation and further experiments were performed at theDepartment of Pharmacy and Biotechnology University of Bologna Bologna Italy Bloodsamples were obtained for patients in control group during the first evaluation by a clinicianTable Cutaneous ulcers were graded using Falanga wound classification systemWound Bed CharacteristicsWound appearanceGranulation tissueFibrinous tissueEscharABCDWound Exudate ScoreExtent of ControlFullyPartiallyUncontrolledAdapted from []101371journalpone0237746t001“ Any amountExudate AmountNoneminimal““““Dressing requirementNo absorptive dressing required If clinically feasibledressings could stay on for up to a weekModerate amountDressing changes required every “ daysVery exudative woundAbsorptive dressing changes required at least dailyPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingT0 and after fifteen days of conventional wound therapy T15 while for HBOT grouppatients before T0 and immediately after the fourth T4 the eighth T8 the twelfth T12and the fifteenth T15 HBOT sessions In addition one month after ending HBOT T1Manother blood sample was collected from HBOT group patients For neutrophil isolationblood was carefully layered on top of an equal volume of Lympholyte1poly Cedarlaneand centrifuged at g for min at “ËšC as previously described [] Plasma wascarefully removed and store at ˚C for further analysis Neutrophils were transferred in aclean tube and were resuspended in mL of HBSS Hanks™ Balanced Salt Solution LifeTechnologies Italia without Ca2Mg2 and centrifuged at g for minutes To lyse theresidual red blood cells RBCs mL Red Cell Lysis Buffer Roche were added and the cellswere resuspended vortexing at low speed to avoid neutrophils activation Neutrophils werecentrifuged at g for min resuspended in HBSS without Ca2Mg2 and adjusted todesired concentrationCell viability was determined using Annexin V7AAD assay Guava Nexin Reagent Millipore as previously described [] and was Differential analysis of cells retrieved usingthis procedure showed granulocytes of which were neutrophils Neutrophilswere stored at room temperature and used for functional tests cell adhesion assay and flowcytometry analysis within h of collection An aliquot of the purified neutrophils was immediately stored at ˚C and used for mRNA extractionNeutrophil adhesion assayAdhesion assays on purified neutrophils were performed as previously described [ ]Briefly black 96well plates were coated overnight at ˚C with fibronectin FN or fibrinogenFg both μgmL to study respectively adhesion mediated by α41 and 2 integrins Neutrophils were counted and stained with CellTracker green CMFDA μM min at ˚CLife Technologies Italia Thereafter cells were plated 50000well on coated wells and incubated for min at ˚C After three washes adhered cells were lysed with Triton X100in BSA bovine serum albumin in HBSS min at ˚C and fluorescence was measuredEx485 nmEm535 nm To evaluate the ability of ligand [] named here RG66 to inhibitneutrophils™ adhesion cells were preincubated with various concentrations ˆ’“ˆ’ M ofRG66 or vehicle methanol for min at ˚C before plating cells into coated wells Neutrophil adhesion assays were also carried out in the presence of an antihuman 2 or α4 integrinantibody both μgmL purified mouse antihuman CD18 and purified mouse antihumanCD49d antibody BD Pharmingen Experiments were carried out in triplicate Data analysisand IC50 values were calculated using GraphPad Prism GraphPad Software San DiegoCA USAFlow cytometry analysisPurified neutrophils were suspended in BSA in HBSS at the concentration of cellsmL μLsample and incubated with FITClabeled antiα4 integrin antibody μLsampleFITC Mouse antihuman CD49d BD Pharmingen or FITClabeled anti2 integrin antibody μLsample FITC Mouse antihuman CD18 BD Pharmingen for min at ˚C as previously described [] After two washes with BSA in HBSS cells were resuspended in PBSand analyzed in a Guava EasyCyte Flow Cytometer Millipore and cellssample wereanalyzed Data were normalized with the relative fluorescence for nonspecific binding evaluated by exposing the cells to an isotype control monoclonal antibody FITC mouse IgG BectonDickinson Italia and set to PLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingIn another set of experiments purified neutrophils were suspended in BSA in HBSS atthe concentration of cellsmL μLsample and incubated with the conformational sensitive phycoerythrin PElabeled HUTS21 monoclonal antibody μLsample PE mouseantihuman CD29 antibody BD Pharmingen for min at room temperature Neutrophilswere washed twice with BSA in HBSS resuspended in PBS and analyzed at the flow cytometry cellssample were analyzed Data were normalized to nonspecific binding relativefluorescence evaluated by exposing the cells to an isotype control mAb monoclonal antibodyand set to Quantitative real time PCRTotal RNA was extracted from purified neutrophils with TRI Reagent SigmaAldrich andquantified using a NanoDrop spectrophotometer ThermoFisher Scientific For each sample“ μg of total RNA was treated with RNasefree DNase as previously described [] TheRNA samples were then converted into cDNA using HighCapacity cDNA Reverse Transcription Kits Life Technologies Italia according to the manufacturer™s instructions RealtimePCR was performed using GoTaq1 qPCR Master Mix Promega Corporation Madison WIUSA The protocol consisted of i for L19 and TNFα denaturation at ˚C for minutesfollowed by cycles of ˚C denaturation seconds and ˚C annealingextension minute ii for α4 integrin denaturation at ˚C for minutes followed by cycles of ˚Cdenaturation seconds ˚C annealing seconds and ˚C seconds iii for IL1denaturation at ˚C for minutes followed by cycles of ˚C denaturation seconds˚C annealing seconds and ˚C seconds Notemplate controls and DNA meltingcurve analysis were used as controls to ensure the lack of contaminating DNA in the RNApreparations and to rule out primerdimer formation respectively To amplify integrin andcytokine targets the following primers were used α4 integrin sense primer 50GTCGCATCCCGTGCAACTTTG30 and antisense primer 50GCTGTGCAGCACGACCGAGT30 amplifying a bp fragment TNFα sense primer 50CTTCTCCTTCCTGATCGTGG30 and antisense primer 50TCTCAGCTCCACGCCATT30 amplifying a bp fragment [] IL1sense primer 50CAAGGGCTTCAGGCAGGCCG30 and antisense primer 50TGAGTCCCGGAGCGTGCAGT30 amplifying a bp fragment [] To amplify 2 integrin cDNA primersequences were from PrimerBank [] a sense primer 50TGCGTCCTCTCTCAGGAGTG30and an antisense primer 50GGTCCATGATGTCGTCAGCC30 amplifying a bp fragmentwere usedAs reference control a bp fragment of the L19 ribosomal protein was amplified using asense primer 50CTAGTGTCCTCCGCTGTGG30 and an antisense primer 50AAGGTGTTTTTCCGGCATC30 [] For data analysis relative expression of RTPCR products wasdetermined using the ΔΔCT method [] as previously described [] the threshold cycle Ctvalues were normalized both on the basis of L19 content and on the values derived from T0sample Each sample was tested in triplicate Primers were synthesized by SigmaAldrichCytokine quantification in plasma by ELISACytokine protein levels TNFα and IL1 were determined in plasma of patients usingELISA kits Invitrogen LifeTechnologies Italia Monza Italy according to the manufacturer™sinstructions Briefly 50μLwell of sample were added to a 96well plate together with μL ofbiotin conjugated primary antibody the plate was then incubated for hours at room temperature After washing four times μL of streptavidinHRP solution were added and the platewas incubated for min at room temperature The wells were washed times and afterwards μL of stabilized chromogen were added and incubated for min at room temperaturePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingAfter the addition of μL of stop solution absorbance was read at nm using an EnSpireMultimode Plate Reader PerkinElmer Waltham MA USA The calculated overall intraassay coefficient of variation was for TNFα and for IL1 the interassay coefficient of variation was calculated to be for TNFα and for IL1Synthesis and bioactivity of α41 synthetic ligandThe synthesis of RRG66 has been previously reported [] via a multistep synthesis startingfrom an enantiopure 3RZtertbutyl 3allylamino2ethylidene4methylpentanoate Thedetailed description of the synthetic protocol has been reported in S1 Fig The biological evaluation showed a strong dependence of the bioactivity on the ring stereochemistry could bedetected since S1 turned out to be completely inactive []Data and statistical analysisAll assays were carried out in triplicate for individual sample at each time pointperson and nrefers to the number of individuals Continuous variables are presented as mean ± standarddeviation when normally distributed data were tested using oneway ANOVA followed byNewmanKeuls posttest or using standard Student t test In addition data are presented asmedian and range and analyzed using MannWhitney™s test when non normally distributedCategorical data were analyzed using χ2square test Data analysis and IC50 values referring toadhesion assays in the presence of RG66 compound were fitted using sigmoidal doseresponseequation using GraphPad Prism software Statistical analyses were performed using GraphPadPrism version GraphPad Software Inc La Jolla CA USA P was consideredsignificantResultsDemographic and clinical data patients men and women age ± years presenting a chronic nonhealingwound condition were recruited and volunteered to participate in the study All the patientscompleted the study and no patient was excluded from the data analysis Demographic parameters such as age gender and clinical data are summarized in Table Age distributions demographic and clinical characteristics of patients were compatiblebetween HBOT and control groups P Overall the wounds of the patients enrolled in the study were caused by different etiologies caused by diabetes by venous insufficiency by critical limb ischemia bytrauma and by vasculitisAs regards comorbidities seven of the subjects suffered from diabetes of type1 and oftype2 twelve of them of hypertension six of obesity seven of venous insufficiency eight ofcardiomyopathy and six of hypothyroidism not requiring thyroid hormones Three subjectswere smokers and four previous smokers Common sites of wound were leg patients foot patients great toe patients and other sites patients Before beginning the HBOTall participants passed a standard medical and physical revision at the Hyperbaric Centre inRavenna All the patients both in control and HBOT groups received standard wound careie wounds were cleaned gently minimizing chemical or mechanical trauma at low pressure“ psi with saline solution and daily sterile sharp debridement with scalpel curette or scissors was performed for the necrotic tissue removal with caution to avoid excess tissue damagewhich may delay healing to get a wellbleeding granulating basePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingPTable Demographic and clinical data of patients enrolled in the studyDemographic dataNumber of subjectsAge yearsaFemaleMalebSmokersComorbiditiesbDiabetesHypertensionObesityVenous insufficiencyGlaucomaCardiomyopathyHypothyroidismHBVHCV positiveRheumatoid arthritisControl groupHBOT group “type1 type2 “ extype1 type2 aAge is expressed as median rangeb Gender and comorbidities are expressed as number of individuals101371journalpone0237746t002Integrin expression on neutrophils deriving from patients undergoingHBOTIn order to study the effect of HBOT on neutrophil integrin expression these cells were isolated from blood samples deriving from patients with a chronic nonhealing wound receiving standard wound care alone control group or undergoing HBOT HBOT group Forcontrol group patients blood samples were collected during the first wound evaluation T0and after fifteen days of conventional wound therapy T15 for patients in the HBOT groupblood samples were obtained before T0 and immediately after the fourth T4 the eighthT8 the twelfth T12 and the fifteenth T15 HBOT treatment at the end of three weeks fiveexposuresweek moreover the last blood sample was drawn one month after ending HBOTT1MAs shown in Fig HBOT did not alter α4 integrin expression in primary human neutrophils both at the mRNA and protein levels Fig panels a and b nor induced any significantvariation in α4 integrin expression throughout the duration of HBO treatment No significantchange in α4 integrin expressed on neutrophils was observed in both control and HBOT grouppatientsOn the contrary 2 integrins were significantly reduced by HBOT both at mRNA andprotein levels this decrement was maintained up to the end of HBO treatment and also onemonth after the last HBOT session Fig panels c and d Neutrophils deriving from patientsreceiving conventional wound care alone control group did not show any changes in 2integrins expressionEffects of HBOT on integrinmediated adhesive properties of neutrophilsDuring an inflammatory process neutrophils adhere and transmigrate through bloodvesselwalls α41 and 2 integrins expressed on neutrophil cell membrane are required and stronglymediate rolling and firm adhesion crawling and transmigration steps of adhesion cascade [] as their activation is an essential step of this complex process To understand whetherPLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT does not modify α4 integrin levels panels a and b but reduces 2 expression panels c and d in neutrophilsderiving from patients receiving standard wound care alone control group and patients undergoing HBOT for sessionsHBOT group The decrement of 2 integrins was maintained up to one month after ending HBOT The effects of HBOT onintegrin expression were evaluated both by qPCR mRNA levels panel a c and by flow cytometry measuring integrin expressed oncell surface panel b d Results from qPCR are expressed as mean ± standard deviation of individual samples carried out intriplicate at each time point control group n HBOT group n Data from flow cytometry analysis are expressed as meanfluorescence intensity MFI ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n MFI values for respective isotype control monoclonal antibody were set to ��� p versus T0both control and HBOT group101371journalpone0237746g001HBOT could influence integrinmediated neutrophil adhesion we performed cell adhesionassays to fibronectin FN or fibrinogen Fg ligands for α41 and 2 integrins respectivelyon neutrophils isolated from patients with chronic nonhealing wound receiving standardwound care alone control group or undergoing HBOT HBOT group Adhesion of neutrophils obtained from patients belonging to both control and HBOT groups was significantlyreduced by the addition of integrin specific antibodies able to block integrin functions Fig demonstrating that neutrophil adhesion to fibronectin or fibrinogen was mainly mediated byα41 or 2 integrin respectively Moreover as shown in Fig exposure to HBO induces a significant reduction of neutrophil adhesion mediated by either 2 or α41 integrins Fig panela and b respectively Interestingly this reduction of neutrophil adhesive properties is retainedthroughout the duration of HBO treatment and up to one month after the last HBOT sessionIn addition these data showed that adhesive properties of α41 integrin expressed on neutrophils were impaired during HBOT although its expression was not modified both at mRNAand protein levels as shown in Fig To better understand the involvement of α41 integrinin neutrophil adhesion we used a conformationspecific antibody that recognizes a specificepitope on integrin 1 subunit exposed only in a defined structural conformation [] Integrins exist in three major conformations a bent or inactive an intermediateactive and an highactivity conformation [] To monitor conformational changes in integrin subunits it is therefore possible to use conformationspecific antibodies [] We employed thePLOS ONE 101371journalpone0237746 August PLOS ONE 0cIntegrins™ role in HBOTpromoted wound healingFig HBOT reduces significantly integrinmediated neutrophil adhesion to fibrinogen Fg or fibronectin FN Integrinmediated adhesion wasdecreased in neutrophils obtained from patients belonging to HBOT group no changes were observed in control group patients The effects of HBOTon neutrophil adhesion mediated by 2 panel a or α41 panel b integrins were evaluated by adhesion assay to Fg or FN respectively as described inmethod section Adhesion mediated by 2 or α41 integrin is significantly prevented in neutrophils treated with a monoclonal antibody anti2 or antiα4 respectively HBOT exposure significantly reduced anti1 HUTS21 mAb binding to neutrophils namely changing α41 integrin conformationpanel c Mean fluorescence intensity MFI due to the anti1 integrin mAb PE conjugated HUTS21 binding in the presence of fibronectin μgmL was measured Nonspecific binding of an isotype control PE conjugated mAb added to neutrophils produced an MFI of ± that was subtractedfrom all samples Data are expressed as mean ± standard deviation of individual samples carried out in triplicate at each time point control groupn HBOT group n �� p � �� p ���� p versus T0 both control and HBOT group101371journalpone0237746g002PEconjugated HUTS21 mAb to determine whether the reduced adhesive properties of neutrophils mediated by α41 integrin during HBOT are due to a conformational change of 1subunit indicative of its activation status HUTS21 antibody recognizes a ligandinducedbinding site that is hidden in the inactive conformation but it is exposed when the agonistbinds or upon partial integrin activation namely when the integrin is in a high affinity conformation The epitope recognized by HUTS21 antibody is located in the hybrid domain of 1integrin subunit [] PEconjugated anti1 HUTS21 mAb was added to neutrophils in thepresence of fibronectin μgmL and fluorescence was measured by flow cytometry Exposure to HBOT induced a significant reduction of HUTS21 mAb binding to neutrophilsthroughout the duration of the treatment and up to one month after the last HBOT sessionFig panel c meaning th
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"carcinogenesis is a process of somatic evolution previous models of stem and transient amplifying cells in epithelial proliferating units like colonic crypts showed that intermediate numbers of stem cells in a crypt should optimally prevent progression to cancer if a stem cell population is too small it is easy for a mutator mutation to drift to fixation if it is too large it is easy for selection to drive cell fitness enhancing carcinogenic mutations to fixation here we show that a multiscale microsimulation that captures both withincrypt and betweencrypt evolutionary dynamics leads to a different epithelial tissues are metapopulations of crypts we measured time to initiation of a neoplasm implemented as inactivation of both alleles of a tumor suppressor gene in our model time to initiation is dependent on the spread of mutator clones in the crypts the proportion of selectively beneficial and deleterious mutations in somatic cells is unknown and so was explored with a parameter when the majority of nonneutral mutations are deleterious the fitness of mutator clones tends to decline when crypts are maintained by few stem cells intercrypt competition tends to remove crypts with fixed mutators when there are many stem cells within a crypt there is virtually no crypt turnover but mutator clones are suppressed by withincrypt competition if the majority of nonneutral mutations are beneficial to the clone then these results are reversed and intermediatesized crypts provide the most protection against initiation these results highlight the need to understand the dynamics of turnover and the mechanisms that control homeostasis both at the level of stem cells within proliferative units and at the tissue level of competing proliferative units determining the distribution of fitness effects of somatic mutations will also be crucial to understanding the dynamics of tumor initiation and progressionk e y w o r d scancer evolution initiation metapopulation dynamics neoplastic progression simulationmajor findings competition between epithelial units such as colonic crypts tends to suppress initiation of neoplasms by suppressing mutator clones this suppression of initiation is enhanced when crypts have few stem cells and so are likely to go extinct due to stochastic fluctuations in stem cell numbers 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 evolutionary applications published by john wiley sons ltdevolutionary applications “ wileyonlinelibrarycom eva 2003 2003 0c 2003 2003 2003 2002 2003 2003introductionthe anization of a population into spatially distinct subpopulations can have a dramatic effect on the evolution of that metapopulation hanski gaggiotti this has implications for both the evolution of anisms and for the effect of tissue architecture on somatic evolution and tissue health in multicellular anisms epithelia are typically divided into subpopulations of tissue stem cells along with the transient amplifying cells and differentiated cells that they produce these subpopulations go by different names in different tissues such as crypts in the intestine or more generally epithelial proliferative units cairns first recognized that the division of stem cells into subpopulations such as crypts acts as a tumor suppressor cairns a mutant stem cell with a reproductive or survival advantage may take over a crypt but is generally constrained from expanding beyond that subpopulation unless it breaches the crypt via a process known as œcrypt fission which tends to duplicate the mutant crypt cell population however by establishing a population size barrier the mutant clone has to overcome the subpopulation structure of the tissue limits the probability that that clone will acquire further carcinogenic mutations yet clones of mutant stem cells can be observed at scales spanning many crypt diameters especially in compromised tissues such as ulcerative colitis and barrett's esophagus maley salk a fundamental question is therefore how the cryptlevel metapopulation dynamics affect the accumulation of somatic mutations during carcinogenesishere we explore the evolutionary dynamics of mutant stem populations that lead to tumor initiation that is the breach of the crypt barrier allowing clonal expansions of crypts across a tissue as well as of mutant stem cells within and out of a crypt while there may be multiple pathways to tumor initiation it has been shown that the inactivation of a single tumor suppressor gene tsg such as the adenomatous polyposis coli apc gene in colon is sufficient to abrogate crypt homeostasis leading to the formation of aberrant crypts and nascent adenomas humphries wright using an agentbased microsimulation model for both stem cell turnover within a crypt and for the cryptpopulation tissuelevel dynamics we study the role of pretumor evolution in tumor initiation this exploration allows for the selection of mutant crypts across the tissue prior to the inactivation of the tumor suppressor gene”a form of premalignant field cancerization”while the stem cell in which the tumor suppressor is inactivated can proliferate beyond the limit of a single crypt due to crypt bifurcationthe evolution of somatic cells is a complex multiscale process depending on the nature of somatic mutations which may either increase or decrease cell fitness stem cell divisions and differentiation or apoptosis as well as subpopulation eg crypt division and extinction rates there is considerable evidence that carcinogenesis involves both an increase in the rate of epigenetic lesions bielas loeb rubin true loeb breivik ji king weisenberger et al and expansions of clones with a relative fitness advantage over their competitor cells cannataro gaffney townsend maley pepper findlay kassen spencer maley vermeulen williams however it continues to be unclear whether the mutator phenotype is a preinitiation phenomenon in carcinogenesis or is more likely to occur during tumor progression in barrett's esophagus another cryptstructured precancer we found evidence that genomic instability precedes genome doubling and transformation martinez the frequency of deleterious versus beneficial mutations in somatic cells is also unknown though the large number of genes in metazoans devoted to differentiation apoptosis and cell cycle control suggests that the frequency of deleterious mutations may be lower in somatic evolution than anismal evolution rajagopalan nowak vogelstein lengauer recent analysis of somatic mutations in cancer found no evidence of purifying selection except in a few essential genes and strong evidence of positive selection with large selective effects williams suggesting that beneficial mutations are more common than deleterious mutations in somatic evolution martincorena although a definitive answer to these questions can only come from further experimental data a theoretical exploration that recognizes the roles of metapopulation dynamics the mutator phenotype and the proportion of deleterious to advantageous mutations in the process of tumor initiation is called for such an exploration will help the identification of factors that drive the tumor initiation processour model integrates previous efforts to characterize the stem cell dynamics within a crypt cannataro mckinley st mary cannataro mckinley st mary frank iwasa nowak komarova komarova cheng loeffler birke winton potten meineke potten loeffler michor frank may iwasa nowak nowak et al pepper sprouffske maley with models of the dynamics of crypt populations cannataro et al chao eck brash maley luebeck kostadinov maley kuhner loeffler bratke paulus li potten totafurno bjerknes cheng mathematical studies of the stem cell population in the crypt niche suggest that epigenetic alterations that increase the rate of genetic lesions mutator mutations and reduce the fitness of stem cells will tend to drift to fixation if the stem cell population is small whereas carcinogenic mutations that increase the proliferation or survival of a stem cell will tend to spread if the stem cell population is large cannataro komarova michor assuming that most nonneutral somatic mutations are deleterious the accumulation of deleterious mutations may lead to senescence of the intestine over time cannataro however competition between crypts of different fitnesses may significantly change the dynamics of the establishment of a mutator clone through a metapopulation dynamic our in silico experiments suggest that there may have been selection at the level of the anism to minimize the number of stem cells within each subpopulation of birtwell 0c 2003 2002 2003 2003its structured epithelium so as to reduce the probability of tumor suppressor gene inactivation and the initiation of carcinogenesisproliferation rate decreased stem cell loss and caused the mutator phenotypethe following equations and assumptions govern the model 2003 2003methodswe implemented a multiscale model of epithelial tissue architecture with stem cells subdivided into crypts under homeostatic control we examined the time required until the two alleles of a tumor suppressor gene tsg were inactivated in at least one stem cell to represent tumor initiation the model was run at least times for every parameter setting crypts were arranged in a flat hexagonal tissue similar to that observed in colon and contained a population of stem cells as well as an implicitly modeled transient amplifying compartment stem cells divided both symmetrically and asymmetrically symmetric division resulted in two daughter stem cells each having the opportunity during the division event synthesis to acquire a mutation asymmetric division did not result in any new stem cells but did provide an opportunity for stem cell mutation stem cell loss due to cell death or differentiation and stem cell gain due to division events were modeled as a stochastic birth“death process with parameters that were functions of the stem cell fitness and of homeostatic feedback effects in response to deviations of the crypt cell population from its normal target level equations and a flow chart of the model algorithm is shown in figure s1homeostasis operated at two spatial scales within a crypt if the stem cell population dropped below the target level stem cell division rates increased by a parameterized amount equation if the population rose above the target level stem cell loss rates increased equation the level of homeostatic feedback was proportional to the degree of deviation away from the target equilibrium level equations and we also introduced a mechanism for homeostasis on the hexagonal lattice of crypts if all the stem cells in a crypt died the inhibition on stem cell population growth was released from the neighboring crypts when the stem cell population of a neighbor reached twice the equilibrium level we modeled crypt bifurcation by allocating half of its stem cells to a new crypt in the location of the dead neighborwe included both beneficial mutations that increased the division probability or the survival probability of stem cells as well as deleterious mutations that decreased them these can accumulate indefinitely and affect fitness multiplicatively equation we also implemented a genetic instability mutation that increased the clone's mutation rate 100fold bielas herr kennedy knowels schultz preston ji king the frequency of each mutation type those that changed the cell's fitness the proportion of nonneutral mutations that were deleterious as well as the rate of tsg inactivation was set by parameters each mutation affected proliferation survival or mutation rate parameters by a constant factor half of the deleterious mutations decreased stem cell proliferation and half decreased their survival increased cell loss for the beneficial mutations increased the cell's 2003 2003equationsequation time to stem cell losslet t be a random exponential deviate with distribution function fr t and rate parameter r the time to cell loss due to apoptosis or differentiation is the minimum of the time to cell loss due to background cell death or differentiation and the time to cell loss due to crypt feedbacktcell_loss min 01t1 ˆ¼ fbackground_loss t2 ˆ¼ ffeedback_loss 01 equation homeostatic crypt feedback by differentiationwhen the stem cell population within a crypt expands beyond the homeostatic target level kcrypt\\_size the crypt provides homeostatic feedback via a change in the rate of stem cell loss with a rate parameter equal to the base stem cell loss rate multiplied by the crypt feedback multiplier the crypt feedback rate multiplier is used to calculate the time to stem cell loss due to crypt homeostatic feedback the crypt feedback multiplier is equal to raised to the nth power where n is the excess number of stem cells above the crypt size divided by the kcrypt\\_deviation parameter here kcrypt\\_deviation and kcrypt\\_size is a parameter that we varied across experimentsrfeedback\\_cell_loss rbase_cell_loss2max0ncellsˆ’kcrypt_sizeˆ•kcrypt_deviationequation homeostatic crypt feedback by proliferationwhen the stem cell population of a crypt drops below kcrypt\\_size the division rate of the remaining stem cells is increased by a factor that depends on the difference between the current number of stem cells ncellsand kcrypt\\_sizerfeedback_division rbase_division2max0kcrypt_sizeˆ’ncellsˆ•kcrypt_deviationequation fitness mutation effectskfitness is a constant factor representing the effect of a single beneficial mutation on fitness as a first approximation we assume that there are many possible mutations that increase and decrease the fitness of a somatic clone by approximately the same amount and so the effect of n beneficial mutations nbeneficial on stem cell fitness is the constant fitness effect raised to the nth power the effect of n deleterious mutations ndeleterious of small effect is just the inverse of kfitness raised to the nth power there is a separate mfitness calculated for the division probability and the survival probability of a cell because beneficial and deleterious mutations may affect either of those probabilitiesmfitness 01kfitness 01nbeneficial 03 kfitness 03ndeleteriousbirtwell 0c 2003 2003 2003 2002f i g u r e 2003plots of cumulative hazard functions using the kaplan“meier estimator the tissue was x crypts with stem cells per crypt in panels a through d each colored line represents the function for a specific proportion of deleterious mutations a baseline experiment with default parameter values table b mutation rate reduced to 01x of baseline c mutator phenotype reduced to 01x of baseline d each colored line represents a different number of cells per crypt all proportions of deleterious mutations were included 2003 2003assumptionscrypts consist of stem cells and of transient amplifying cellscrypt density is fixed that is the tissue contains a fixed number of crypts arranged on a hexagonal griddrops below the target level the division rate of each stem cell in the crypt is increased when the number of stem cells grows above the target level the cell loss rate of each stem cell in the crypt is increasedcrypts divide to fill vacant slots left by adjacent crypts that have the number of cells in a crypt transient amplifying compartment gone extinct due to loss of the constituent stem cellsis fixedcrypts attempt to maintain a stable population of stem cells through homeostatic feedback when the number of stem cells the extinction of an adjacent crypt suppresses the homeostatic apoptotic signals allowing the stem cell populations in neighboring crypts to expand once that extinct crypt is replaced the normal birtwell 0cta b l e 2003baseline simulation parameterssimulationmaximum simulation duration in daysstem celldivision rate rbase\\_divisionratio of asymmetric divisions to symmetric divisionsstem cell loss rate rbase\\_cell\\_lossmutation rate per stem cell divisionmutation rate maximumtumor suppressor gene mutation ratetumor suppressor gene mutation rate maximumnumber of transient amplifying cells associated with each stem cellcell division minimum time in dayscell loss minimum time in dayscrypttarget equilibrium level of number of stem cells in a crypt kcrypt\\_sizestandard deviation from equilibrium level the crypt uses this value to determine its level of effect on the cell loss and division rates of the stem cells kcrypt\\_deviationbifurcation threshold factor below which a crypt will not bifurcatecrypt cell loss effect multipliercrypt division effect multipliermultiplier to the division effect for each dead neighbor cryptcanceruncontrolled cell proliferation threshold if a crypt has this threshold times the equilibrium number of stem cells it is considered to be experiencing uncontrolled stem cellnumber of tumor suppressor gene hits that mean cancermutation 2003 2002 2003 2003 years x “ x “ x “ also tested x “ x “default varied from “percent of nonneutral mutations that are deleteriousthe factor affecting the loss rate of the stem cell from a beneficial mutation ˆ•kfitnessthe factor affecting the division rate of the stem cell from a beneficial mutation kfitnessthe factor affecting the cell loss rate of the stem cell from a deleterious mutation kfitnessthe factor affecting the division rate of the stem cell from a deleterious mutation ˆ•kfitnessthe factor affecting the mutation rate of the stem cell from a mutator mutation“ also tested homeostatic controls on stem cell numbers of neighboring crypts are restoredcrypt division is triggered by an expansion of the stem cell population of a crypt to twice its homeostatic level as hypothesized by garcia park novelli and wright as long as there is an empty slot adjacent to the enlarged crypta stochastic birth“death process governs the scheduling of division and cell loss eventsfitness mutations affect in a multiplicative fashion the rate parameters of the birth“death processthere is a single mutator phenotype that requires only a single mutator mutation additional mutator mutations have no effect on the mutation ratethe loss of the first allele of the tsg has no effect on stem cell fitness 2003 2003results 2003 2003tsg inactivation depends on the emergence of a mutatorat baseline for comparison our tissue was a 5x5 hexagonal lattice of crypts each crypt having stem cells stem cell loss and symmetric division rates were balanced mutations were acquired stochastically with probabilities defined by proportions starting with deleterious mutations beneficial mutations and mutator mutations and ranging in increments to deleterious beneficial and mutator beneficial versus mutator the incidence of tsg inactivation decreased as the proportion of deleterious mutations increased figure 1a table birtwell 0c 2003 2003 2003 2002reducing the base mutation rate to of baseline we observed a marked decrease in tsg inactivation figure 1b as expected similarly reducing the effect of the mutator mutation to 10x the baseline mutation rate instead of 100x produced a significant decrease in tsg inactivation figure 1c we found that the vast majority of tsg inactivation occurred in stem cells that had previously acquired the mutator phenotype figure s2 this assumes that the mutator phenotype can be caused by a single mutation that is otherwise neutral eg overexpression of dna polymerase beta canitrot or a dominantnegative mutation in p53 de vries though this assumption is easily relaxed 2003 2003proportion of deleterious mutations negatively correlates with tsg inactivationnot surprisingly we found that the proportion of deleterious mutations and the incidence of tsg inactivation were negatively correlated figure cell divisions per time remained roughly constant across all proportions of mutations however as the proportion of deleterious mutations decreased the cost of being a mutator also decreased because it accumulated less mutational burden of deterious mutations this resulted in an increased emergence of crypts with fixed mutator stem cell populations conversely across all experiments we observed progressively less tsg inactivation as the proportion of deleterious mutations approached our maximum of turnover levels we observed a reduction in the average number of stem cells per crypt figure s3 the implemented homeostatic control was unable to maintain the target stem cell population size in the face of high turnover rates essentially there is a lag between depletion of the stem cell pool due to cell death and differentiation and replenishment provided by an increase in stem cell division rates with higher levels of cell loss the simulated crypts spend more time further below the target homeostatic number of stem cells as a result there were fewer total stem cells in the simulation and therefore fewer mutations per time allowing less chance for mutator acquisition and tsg inactivation this may or may not be realistic second as the turnover level increased above our baseline the number of mutator crypts present in the tissue at any given time decreased figure since increased turnover should lead to increased opportunities for mutator mutations to arise the decline in mutator crypts was a surprise however the loss of mutator crypts is due to intercrypt competition as described below increased turnover led to increased stochastic fluctuations in stem cell numbers and thereby increased crypt extinction events the reduction in stem cell numbers and mutator crypts combined to produce a reduction in the overall incidence of tsg inactivation as turnover rates increased above baseline if in reality homeostatic control of stem cell numbers prevents increased crypt extinctions with increased stem cell turnover this result would likely not hold however all things being equal increased cell turnover would be expected to increase stem cell number fluctuations 2003 2003increased stem cell turnover initially increased and then decreased tsg inactivationwe modulated stem cell turnover by varying cell loss and symmetric division rates in unison at lower turnover levels we found that increased cell turnover increased tsg inactivation however at turnover levels 2x and 5x our baseline level the incidence of tsg inactivation declined figure due to two factors first at higher 2003 2003the number of stem cells per crypt had a varying effect on tsg inactivationin general fewer stem cells per crypt reduced the rate of tsg inactivation even though the total number of stem cells in the tissue was held constant figure 1d however there is a tradeoff between tsg inactivation and tissue death at very low stem cells per crypt with only one stem cell per crypt there is no opportunity for homeostatic signals within a crypt to compensate for stem cell loss in f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed each bar corresponds to a specific proportion of deleterious mutations the proportion of mutator crypts correlates with the risk of tumor initiation as seen in figure birtwell 0cbase cell loss divison rate base cell loss divison rate 2003 2002 2003 2003base cell loss divison rate base cell loss divison rate f i g u r e 2003the effect of changes in stem cell turnover plots of cumulative hazard functions using the kaplan“meier estimator where each colored line represents the function for a specific proportion of deleterious mutations the baseline division and stem cell loss rates were as seen in figure initially turnover correlated positively with the risk of tumor initiation however at higher turnover rates the risk of tumor initiation decreased due to a reduction in the overall number of living stem cells and decreased incidence of mutator cryptsour model tissues with one stem cell per crypt were mostly unviable and died out before tsg inactivation or the predetermined simulation end timeat and cells per crypt we observed a reduction in the incidence of tsg inactivation figure 1d at all but the lowest proportions of deleterious mutations as was predicted by models of the crypt stem cell niche of single crypts komarova michor figure s4 as in other experiments along with a reduction in the incidence of tsg inactivation the frequency of fixed mutator crypts was reduced as well this shows that selection against mutator cells increases as the number of stem cells increases above some threshold in our model as long as the majority of nonneutral mutations are deleterious supporting the s by michor and komarova komarova michor et al when the stem cell populations are large it is very unlikely that a crypt will go extinct and so there is no intercrypt competition in this case the metapopulation dynamics are reduced to the single crypt dynamicsthe increased risk of tsg inactivation associated with increased stem cells per crypt appeared to plateau after approximately stem cells per crypt the total number of cells in the tissue remained constant as did the total stem cell divisions per time figure s5 the average number of mutations per time increased through stem cells per crypt but then reached a temporary plateau figure there was no statistically significant difference between the average number of mutations per time in the and stem cells per crypt cases as the number of stem cells per crypt increased beyond the average mutations per time decreased except when the proportion of deleterious mutations was the incidence of mutator crypts followed a similar trend figure birtwell 0c 2003 2003 2003 2002f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed as a function of turnover rate each bar corresponds to a specific proportion of deleterious mutations as turnover increased mutator crypts became more rare at higher proportions of deleterious mutationsf i g u r e 2003number of mutations per unit time as a function of cells per crypt and proportion of deleterious mutations where each bar represents a specific proportion of deleterious mutations at most proportions of deleterious mutations mutations per time peaked around stem cells per crypt at deleterious mutations mutations per time reached a minimum at cells per crypt and increased as the cells per crypt grew past we have chosen to explore the case where the total number of stem cells is kept constant assuming that a certain number of selfrenewing tissue stem cells might be required to maintain an epithelial tissue an alternative view is that a fixed number of epithelial units like the crypts might be required to maintain a tissue and that the number of stem cells per crypt could vary by changing the number of differentiated cells produced by each stem cell in this case the risk of tsg inactivation continues to increase with increasing number of stem cells per crypt figure s4 because we held the number of crypts constant but changed the number of cells per crypt in this case the total number of cells in this simulation also changed leading to a large evolving population size of stem cells and thus an increased chance for at least one cell to inactivate both alleles of the tsg 2003 2003partitioning the tissue into crypts imposed a metapopulation dynamicfitness levels measured by the difference between division and loss rates were greater in tissues with smaller crypts even as the total number of stem cells remained constant figure 6a counts of crypt births and crypt life span measurements showed that there was more crypt turnover in smaller crypts figures s6 and s7 this crypt turnover provided an opportunity for fitter phenotypes to spread more easily across the tissue crypt fitness peaked at cells per crypt where there was the most crypt turnover and bottomed out at and cells per crypt after cells per crypt we observed no crypt death and therefore no turnover crypt fitness began to increase again at and cells per crypt through natural selection of stem cells within larger crypts however fitness levels did not increase to the levels seen at cells per cryptthe metapopulation dynamic was observed in the clonal expansion of mutator crypts across the tissue we considered two crypts to have œmutator phenotype agreement if they both have a fixed mutator mutation or neither has a fixed mutator we calculated mutator crypt agreement across spatial distance and found that overall agreement decreased as the cells per crypt increased figure 6b further at and cells per crypt closer crypts had increased mutator agreement suggesting clonal expansion of mutator crypts conversely at cells per crypt and above we found that spatial distance correlated less well with mutator agreement indicating that birtwell 0c 2003 2002 2003 2003f i g u r e 2003a crypt fitness measured by the difference between division and cell loss rates across cells per crypt and proportion of deleterious mutations the total number of stem cells remained constant across these experiments the crypt turnover at and cells per crypt allowed fit clones to spread across the tissue resulting in increased overall fitness b mutator agreement by distance class two crypts were in œmutator agreement if they both had fixed mutator mutations or neither did overall agreement was higher in the smaller crypts suggesting that mutator clones were able to spread across the tissuewhen there was less crypt turnover mutator crypts arose de novo rather than through cryptlevel clonal expansion 2003 2003discussionin the colon the development of adenomatous polyps frequently involves the inactivation of the apc gatekeeper gene a member of the wntsignaling pathway which represses proliferation and facilitates orderly cell differentiation in the luminal part of the crypts barker goss groden as long as this gatekeeper gene is active mutant stem cell progeny with neoplastic potential is likely eliminated from the crypt and clonal expansion thus averted however when the gatekeeper gene is inactivated the brakes on mutant stem proliferation are removed and mutant cell progeny may undergo focal clonal expansion therefore we asked the question what are the factors that determine the risk of a tsggatekeeper inactivation leading to tumor initiation in a compartmentalized tissue a metapopulationour microsimulations indicate that the base mutation rate figure 1b and the increase in that rate for a mutator clone figure 1c are the main driving forces behind tsg inactivation and thus the initiation of carcinogenesis the proportion of deleterious mutations is important for its effect on selection of the mutator clone we find that if most mutations are assumed to be deleterious then a mutator clone quickly accumulates a large genetic load of deleterious mutations and tends to be driven to extinction in competition with nonmutator cells however if mutations are more likely to be beneficial then a mutator clone can spread more easily which leads to the early inactivation of both alleles of the tumor suppressor gene figure 1a similarly increasing turnover of the stem cells effectively increases the mutation rate and consequently the rate of initiation however when turnover is very high the homeostatic feedback signals cannot maintain the target number of stem cells and so the total stem cell population of the epithelium decreases which in turn decreases the chances for initiation figure since the rate of tsg inactivation is trivially relat
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" This delay was largely influenced by the interests of Metropolitan Life Insurance Company (MetLife) and other asbestos mining and product manufacturing companies. Objectives: To understand the ongoing corporate influence on the science and politics of asbestos and silica exposure including litigation defense strategies related to historical manipulation of science. Methods: We examined previously secret corporate documents depositions and trial testimony produced in litigation; as well as published literature. Results: Our analysis indicates that companies that used and produced asbestos have continued and intensified their efforts to alter the asbestos“cancer literature and utilize dust-exposure standards to avoid liability and regulation. anizations of asbestos product manufacturers delayed the reduction of permissible asbestos exposures by covering up the link between asbestos and cancer. Once the decline of the asbestos industry in the US became inevitable the companies and their lawyers designed the state of the art (SOA) defense to protect themselves in litigation and to maintain sales to developing countries. Conclusions: Asbestos product companies would like the public to believe that there was a legitimate debate surrounding the dangers of asbestos during the twentieth century particularly regarding the link to cancer which delayed adequate regulation. The asbestos“cancer link was not a legitimate contestation of science; rather the companies directly manipulated the scientific literature. There is evidence that industry manipulation of scientific literature remains a continuing problem today resulting in inadequate regulation and compensation and perpetuating otherwise preventable worker and consumer injuries and deaths. asbestos mesothelioma state-of-the-art corporate corruption MetLife industry knowledge 9421547 4136 Hum Pathol Hum. Pathol. Human pathology 0046-8177 1532-8392 24746212 4271837 10.1016/j.humpath.2014.01.003 NIHMS648391 Y-chromosome status identification suggests a recipient origin of posttransplant non“small cell lung carcinomas: chromogenic in situ hybridization analysis??? Chen Wei MD PhD a Brodsky Sergey V. MD PhD a Zhao Weiqiang MD PhD a Otterson Gregory A. MD b Villalona-Calero Miguel MD b Satoskar Anjali A. MD a Hasan Ayesha MD b Pelletier Ronald MD c Ivanov Iouri MD PhD a Ross Patrick MD PhD c Nadasdy Tibor MD a Shilo Konstantin MD a * aDepartment of Pathology The Ohio State University Wexner Medical Center Columbus OH 43210 bDepartment of Medicine The Ohio State University Wexner Medical Center Columbus OH 43210 cDepartment of Surgery The Ohio State University Wexner Medical Center Columbus OH 43210 *Corresponding author. Department of Pathology The Ohio State University Wexner Medical Center E412 Doan Hall 450 West 10th Ave Columbus OH 43210. Konstantin.ShiloOSUMC.edu (K. Shilo) 13 12 2014 23 1 2014 5 2014 19 12 2014 45 5 1065 1070 2014 Elsevier Inc. All rights reserved. 2014 Summary Owing to the need of lifelong immunosuppression solid-an transplant recipients are known to have an increased risk of posttransplant malignancies including lung cancer. Posttransplant neoplastic transformation of donor-derived cells giving rise to hematopoietic malignancies Kaposi sarcoma and basal cell carcinoma in nongraft tissues has been reported. The goal of this study was to assess the cell origin (donor versus recipient derived) of posttransplant non“small cell lung carcinomas (NSCLCs) in kidney and heart transplant recipients. An institutional database search identified 2557 kidney and heart transplant recipients in 8 consecutive years. Among this cohort 20 (0.8%) renal and 18 (0.7%) heart transplant recipients developed NSCLC. The study cohort comprised 6 of 38 NSCLCs arising in donor-recipient sex-mismatched transplant patients. The tumor cell origin was evaluated by chromogenic in situ hybridization with Y-chromosome probe on formalin-fixed paraffin-embedded tissues. Y-chromosome was identified in 97% ± 1% (range from 92% to 99%) of all types of nucleated cells in male control tissues. In all 5 NSCLCs from male recipients of female donor an Y-chromosome was identified in 97% ± 2% (range from 92% to 100%) of tumor cells statistically equivalent to normal control (P < .001). No Y-chromosome was identified in NSCLC cells from a female recipient of male kidney. These findings suggest a recipient derivation of NSCLC arising in kidney and heart transplant recipients. A combination of histologic evaluation and chromogenic in situ hybridization with Y-chromosome analysis allows reliable determination of tissue origin in sex-mismatched solid-an transplant recipients and may aid in management of posttransplant malignancy in such cases. Post“solid-an transplantation lung cancer Chromogenic in situ hybridization for Y-chromosome 15030100R 648 Ann Thorac Surg Ann. Thorac. Surg. The Annals of thoracic surgery 0003-4975 1552-6259 24576597 4008142 10.1016/j.athoracsur.2013.12.043 NIHMS571118 Accuracy of FDG-PET within the clinical practice of the ACOSOG Z4031 trial to diagnose clinical stage I NSCLC Grogan Eric L. MD MPH a b c Deppen Stephen A. MA MS PhD b c * Ballman Karla V. f Andrade Gabriela M. b Verdial Francys C. b Aldrich Melinda C. b d Chen Chiu L. e Decker Paul A. f Harpole David H. MD g Cerfolio Rrobert J. MD h Keenan Robert J. MD i Jones David R. MD j D™Amico Thomas A. MD g Shrager Joseph B. MD k Meyers Bryan F. MD l Putnam Joe B. Jr. MD a b aVeterans Affairs Medical Center Nashville TN bDepartment of Thoracic Surgery; Department of Medicine Vanderbilt University Medical Center Nashville TN cInstitute for Medicine and Public Health Vanderbilt University Medical Center Nashville TN dDivision of Epidemiology Vanderbilt University Medical Center Nashville TN eCenter for Quantitative Sciences Vanderbilt University Medical Center Nashville TN fDivision of Biomedical Statistics and Informatics Mayo Clinic Rochester MN gDepartment of Surgery Duke University Durham NC hDepartment of Surgery University of Alabama Birmingham AL iDepartment of Surgery Allegheny General Hospital Pittsburgh PA jDepartment of Surgery University of Virginia Charlottesville VA kDepartment of Surgery Stanford University Stanford CA lDepartment of Surgery Washington University St. Louis MO Corresponding Author/Request for Reprints: Eric Grogan M.D. M.P.H. Department of Thoracic Surgery 609 Oxford House 1313 21st Ave. South Nashville TN 37232 Phone: 615-322-0064 Fax: 615-343-9194 eric.groganvanderbilt.edu * Equal shared co-first author 23 4 2014 25 2 2014 4 2014 01 4 2015 97 4 1142 1148 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved. 2014 Background Fluoro-deoxyglucose positron emission tomography (FDG-PET) is recommended for diagnosis and staging of NSCLC. Meta-analyses of FDG-PET diagnostic accuracy demonstrated sensitivity and specificity of 96% and 78% respectively but were performed in select centers introducing potential bias. This study evaluates the accuracy of FDG-PET to diagnose NSCLC and examines differences across enrolling sites in the national ACOSOG Z4031 trial. Methods 959 eligible patients with clinical stage I (cT1-2N0M0) known or suspected NSCLC were enrolled between 2004 and 2006 in the Z4031 trial and 682 had a baseline FDG-PET. Final diagnosis was determined by pathological examination. FDG-PET avidity was categorized into four levels based on radiologist description or reported maximum standard uptake value (SUV). FDG-PET diagnostic accuracy was calculated for the entire cohort. Accuracy differences based on preoperative size and by enrolling site were examined. Results Preoperative FDG-PET results were available for 682 participants enrolled at 51 sites in 39 cities. Lung cancer prevalence was 83%. FDG-PET sensitivity was 82% (95% CI: 79“85) and specificity was 31% (95% CI: 23%“40%). Positive and negative predictive values were 85% and 26% respectively. Accuracy improved with lesion size. Of 80 false positive scans 69% were granulomas. False negative scans occurred in 101 patients with adenocarcinoma being the most frequent (64%) and eleven were ?10mm. The sensitivity varied from 68% to 91% (p=0.03) and the specificity ranged from 15% to 44% (p=0.72) across cities with > 25 participants. Conclusions In a national surgical population with clinical stage I NSCLC FDG-PET to diagnose lung cancer performed poorly compared to published studies. Tumour Biol Tumour Biol Tumour Biology 1010-4283 1423-0380 Springer Netherlands Dordrecht 24510347 4053595 1674 10.1007/s13277-014-1674-x Research The diagnostic and prognostic value of serum human kallikrein-related peptidases 11 in non-small cell lung cancer Xu Chun-Hua Zhang Yu Yu Li-Ke +86-25-8372-8558 +86-25-83728558 yulike_doctor163.com Department of Respiratory Medicine Nanjing Chest Hospital 215 Guangzhou Road Nanjing 210029 China Nanjing Clinical Center of Respiratory Diseases Nanjing China 9 2 2014 9 2 2014 6 2014 35 6 5199 5203 6 12 2013 22 1 2014 The Author(s) 2014 Open Access This is distributed under the terms of the Creative Commons Attribution License which permits any use distribution and reproduction in any medium provided the original author(s) and the source are credited. The aim of this study was to explore the diagnostic and prognostic value of serum human kallikrein-related peptidases 11 (KLK11) level in non-small cell lung cancer (NSCLC). Serum specimens from 138 patients with NSCLC and 40 healthy controls were collected. The concentration of KLK11 was measured by enzyme-linked immunosorbent assay (ELISA). The concentration of KLK11 in NSCLC was significantly higher compared to that in the controls (P?<?0.01). The serum KLK11 levels decreased with stage presence of lymph node and distant metastases regardless of histology age and sex. With a cutoff point of 1.05 ng/ml KLK11 showed a good diagnostic performance for NSCLC. Univariate analysis revealed that NSCLC patients with serum high KLK11 had a longer overall survival (OS) and progression-free survival (PFS) than those with low KLK11 (HR of 0.36 P?=?0.002; HR of 0.46 P?=?0.009). Cox multivariate analysis indicated that KLK11 was an independent prognostic indicator of PFS and OS (HR of 0.53 P?=?0.042; HR of 0.48 P?=?0.037). Kaplan“Meier survival curves further confirmed that patients with high KLK11 have longer PFS and OS (P?=?0.003 and P?=?0.018 respectively). In conclusion the measurement of KLK11 might be a useful diagnostic and prognostic test for NSCLC patients. Keywords Kallikrein-related peptidases 11 Non-small cell lung cancer Diagnosis Prognosis issue-copyright-statement International Society of Oncology and BioMarkers (ISOBM) 2014 Introduction Lung cancer is the leading cause of cancer-related death worldwide with more than 1.2 million deaths each year [1]. Non-small cell lung cancer (NSCLC) accounts for 80“85 % of total lung malignancies [2]. Although advances in noninvasive methods have improved our ability to detect lung cancer more than 75 % of lung cancer patients present an advanced stage of disease [3] and they have little prospect of effective and curative treatment with 5-year survival rates of less than 15 % [4]. Tumor markers play a key role in patient management for many malignancies. The potential uses of serum tumor markers include aiding early diagnosis determining prognosis prospectively predicting response or resistance to specific therapies and monitoring therapy in patients with advanced disease. Kallikrein-related peptidases 11 (KLK11) is a member of the human kallikrein gene family which localized on chromosome 19q13.4 [5]. Recent studies have reported that KLK11 has been expressed in many cancers including prostate cancer [6] ovarian cancer [7] gastric cancer [8] as well as rectal carcinoma [9]. An immunofluorometric assay study demonstrated that KLK11 expression in ovarian cancer tissues is a marker of favorable prognosis since patients with KLK-positive tumors exhibit a longer progression-free survival (PFS) and overall survival (OS) [10]. Additionally Sasaki et al. [11] reported that lower KLK11 mRNA expression in lung cancer is an indicator of poor prognosis in patients with lung cancer. However there seems to be a paucity of research concerned with serum KLK11 expression in NSCLC. For this reason the goal of the present study was to investigate the baseline serum levels of KLK11 in patients with NSCLC to determine its potential diagnostic and prognostic roles. Materials and methods Patients A total of 138 patients with NSCLC were examined at the Nanjing Chest Hospital between January 2006 and May 2008. The cohort of patients included 80 (58.0 %) male and 58 (42.0 %) female subjects with a median age of 56 years (range 45“68 years). The clinical features of the patients are summarized in Table 1. Follow-up lasted through December 2012 with a median follow-up period of 22 months for living patients (range 3“80 months). PFS was defined as the time interval between the date of diagnosis and the date of disease relapse. OS was defined as the time interval between the date of diagnosis and the date of death.Table 1Clinical characteristics of NSCLC patients and controlsVariablesNSCLCControl P valueSubject no.13840Age year57.8?±?10.254.6?±?7.80.614Male/Female80/5826/140.325Histology?AC78?SCC60 AC adenocarcinoma SCC squamous cell carcinoma The diagnosis of lung cancer was made using various methods: sputum cytology fine-needle aspiration or bronchoscopy as dictated by the patient™s presentation. Pathologists interpreted the cytology or histology of tissue biopsy. Lung cancer was staged using a widely used classification system and the staging procedure included a clinical examination; CT of the chest abdomen and brain; abdominal ultrasonography; bone scanning; and positron emission tomography. The study protocol was approved by the ethics committee of Nanjing Chest Hospital. All patients provided written informed consent before enrollment. Measurement of serum KLK11 levels Serum samples from each individual were obtained at the time of diagnosis before any therapeutic measures were started (surgery chemotherapy or radiation). Samples were centrifuged at 1500×g for 10 min at ?4 °C. The supernatant was stored at ?80 °C for assessment of the levels of KLK11. The KLK11 concentration was determined by ELISA with the commercial KLK11 ELISA Ready-SET-Go kit (eBioscience San Diego CA). All samples were blinded to the technologists running the assays and the code was broken to the statisticians after the database was constructed. Statistical analysis Statistical software (SPSS for Windows version 18) was used for the analysis. Differences between independent groups were examined by the Mann“Whitney U test. To determine the diagnostic accuracy of KLK11 receiver operating characteristic (ROC) curves were retrieved from logistic regression analysis and the area under the curve (AUC) was calculated. Univariate survival analysis was performed using the Kaplan“Meier method and the log-rank test. Multivariate analysis was conducted to determine an independent impact on survival using the Cox proportional hazard method. P?<?0.05 was considered statistically significant. Results Comparison of serum KLK11 levels between NSCLC patients and controls As shown in Fig. 1 the concentration of KLK11 was significantly higher in patients with NSCLC (2.04?±?0.86 ng/ml) than in those with the controls (0.93?±?0.52 ng/ml) (P?<?0.01).Fig. 1Levels of KLK11 in NSCLC. Among 138 NSCLC patients the serum levels of KLK11 were 2.04?±?0.86 ng/ml which were significantly higher than 0.93?±?0.52 ng/ml in healthy controls (P?<?0.01) Diagnostic value of KLK11 in NSCLC A ROC curve analysis was carried out to assess the value of KLK11 in NSCLC. The area under the ROC curve was 0.892 (confidence interval (95 % CI) 0.841“0.942). With a cutoff point of 1.05 ng/ml which was defined as the normal value based on the mean value plus two standard deviation obtained from healthy controls serum KLK11 has a sensitivity of 65.9 % (91/138) a specificity of 82.5 % (33/40) an accuracy of 69.7 % (124/178) a positive predictive value of 92.9 % (91/98) and a negative predictive value of 41.3 % (33/80) (Fig. 2).Fig. 2ROC of KLK11 for the diagnosis of NSCLC. Serum levels of KLK11 among 138 NSCLC patients and 40 healthy controls were determined. The diagnostic potentials of KLK11 were "
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"The VATS approach is a safe and feasible treatment in terms of the survival rate for metastatic lung cancer compared with the thoracotomy. The 3-year disease-free survival rate in the VATS group is inferior to that of open thoracotomy. The VATS approach could not completely replace open thoracotomy. The authors have no support or funding to report. Introduction Metastasectomy is considered a beneficial treatment for a patient with metastatic lung cancer whose primary tumor has been well controlled[1].After surgery 5-year survival rates of 30% to 50% could be achieved depending on the underlying primary cancer[2]“[4].In practice the surgical approaches to pulmonary metastases are variable. Video-assisted thoracoscopic surgery (VATS) is an emerging technique; many procedures that had previously required a thoracotomy have been performed with the minimally invasive VATS. VATS has been used for the treatment of pulmonary metastases. The routine use of VATS for the treatment of respectable metastatic lung cancer remains controversial. Critics of the VATS approach have argued that it might not be an equivalent oncological operation[5] [6]. A prospective study by Cerfolio[7]found that 22% of the nodules that could be detected by thoracotomy were missing by VATS.Whether the VATS approach can provide a satisfactory outcome is unknown. An evidenced-based investigation of the VATS approach is needed we undertook this meta-analysis to achieve a more objective assessment of the published studies and to provide a more accurate comparison between VATS and thoracotomy for metastatic lung cancer. Methods Search Strategy Electronic searches were of the MEDLINECochrane Controlled Trial Register (CENTRAL) Ovid MEDILINE PubMed and Embase databases were performed until July 2013.The following MeSH search headings were used: œmetastatic lung cancer œpulmonary metastases œvideo-assisted thoracic surgery œthoracotomy and œcomparative study.We searched the reference lists of relevant studies reviews editorials lettersand meeting s. We used the Science Citation Index to cross-reference for further studies that met our criteria. Study Selection The studies included in this meta-analysis were based on our predetermined criteria as follows: (1) clinical trials that include the full text of the paper published in peer-reviewed English journals or reports of presentations at major thoracic surgery meetings; (2) comparison of the efficacy of VATS to that of thoracotomy in patients with metastatic lung cancer; and (3) similarity in the patients' baseline characteristics. Data extraction and quality assessment Two independent reviewers (Siyuan and Wenya) assessed the quality and the risk of bias of the included trials as follows: (1) the studies that did not include a comparative group with surgery as a form of intervention were excluded; (2) the trials focusing on patients undergoing surgery for primary lung cancer were excluded; (3) the studies on robotic video-assisted thoracic surgery were excluded; (4) if there was an overlap between authors centers or patient cohorts evaluated in the published literature only the most recent report was included; (5) studies published more than 20 years ago were excluded because of the significant technological changes that has occurred. The s were evaluated with the Downs and Black quality assessment method[8]. Discrepancies between the two investigators were resolved by discussion and consensus with a senior investigator. The final results were reviewed by two senior investigators (Lin and Jiang).The disease-free survival was defined as the date of the initial metastasectomy until the date of a recurrence. Statistical and sensitivity analyses The meta-analysis was performed using the RevMan 5.1.0. software package. The odds ratio (OR) or the mean difference with 95% confidence intervals (95% CI) was calculated for the dichotomous outcomes and the continuous outcomes respectively. A P value<0.05 was considered a significant difference in the value between the two groups. We used the I2 statistic to investigate the heterogeneity among the studies.The heterogeneity was explored by X2 and I2; I2<25% and I2>50% reflect a small and large inconsistency respectively. P<0.05 was considered significant. If there were a statistical difference in terms of the heterogeneity (P?0.05) a random-effect model was selected to pool the data. Otherwise a fixed-effect model was used. Taking into account the presence of different sample sizes of the included studies a sensitivity analysis was performed to compare the of 1-year survival rate and the 3-year disease free survival rate between VATS and open thoracotomy. Publication bias A funnel plot was used to explore bias. Asymmetry in the funnel plot of trial size against treatment effect was used to assess the risk of bias. Results Description of the studies Six retrospective cohort studies the met our criteria were included in this meta-analysis. A total of 546 patients were included in the six studies;235 patients were allocated to the VATS group whereas 311 were allocated to the open thoracotomy group to evaluate their survival rate.The search algorithm results of the search strategies and selection criteria are shown in Fig 1. The patient characteristics and evaluation index are shown in . .0085329.g001 Identification of studies for inclusion. .0085329.t001 Study Design Country NO(V/O) Gender (M/F) Mean age (years) Assessment score Nakajima2001[28] OC Japan 45/55 V59/41 O34/21 V55±15 O55±14 13 Mutsaerts2002[29] OC Netherlands 8/12 NR NR 19 Nakas2009[30] OC UK 25/27 V16/9 O 19/8 V69 O66 16 Carballo2009[31] OC USA 36/135 V18/18 O82/53 V58.5 O49 15 Gossot2009[32] OC France 31/29 V21/10 O13/16 V43 O40 18 Chao2012[33] OC Taiwan 90/53 V49/41 O35/18 NR 13 V VATS; O Open thoracotomy; NR Not reported; OC observational cohort. Assessment of Recurrence and Survival Six studies documented the 1-year survival rateand there was no significant heterogeneity among the six studies (x2?=?3.79 P?=?0.58I2?=?0%).A fixed effect model was used.The combined result is shown in Fig 2(OR?=?1.15; 95%CI 0.72“1.84; p?=?0.58). Because of the heterogeneity in sample size the sensitivity analyses were conducted using larger sample sizes. There was no difference between the two surgical methods with an OR of 1.00(95%CI 0.55“1.79) and with heterogeneity(?2?=?3.23P?=?0.07 I2?=?69%). Five studies reported the 3-year survival rate and heterogeneity was identified through the five studies (x2?=?11.32P?=?0.02I2?=?65%); and a random effect model was adopted (OR?=?1.07; 95%CI 0.50“2.27; p?=?0.86) (Fig 3). Three studies compared the 5-year survival rate (OR?=?0.96; 95%CI 0.34“2.71; p?=?0.93) with certain heterogeneity(x2?=?8.86P?=?0.01I2?=?77%) (Fig 4). .0085329.g002 1-year survival rate. Forest plot of the Odds Ratio(OR) of the 1-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g003 3-year survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g004 5-year survival rate. Forest plot of the Odds Ratio(OR) of the 5-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. Four studies compared the 1-year disease free survival rate (OR?=?1.31; 95% CI 0.79“2.19; p?=?0.30)finding no significant heterogeneity among these studies (x2?=?1.82P?=?0.61I2?=?0%) (Fig 5) and four studies compared the 3-year disease free survival rate (OR?=?0.59; 95% CI0.38“0.91; p?=?0.02) finding no significant heterogeneity (x2?=?1.82P?=?0.61I2?=?0%) between the patients who underwent VATS and those who underwent open thoracotomy (Fig 6). Because of the heterogeneity in the sample size sensitivity analyses were conducted using larger sample size studies; however there was no difference between the two surgical methods with an OR of 1.71 (95% CI1.02“2.89) and with heterogeneity (?2?=? 3.07P ?=?0.22 I2?=?35%). There were significant 3-year disease free survival rate benefits with open thoracotomy. We attempted to evaluate the 5-year disease free survival rate.Only two studies reported these ratesand the published data were not sufficient for the combined analysis. .0085329.g005 Figure 5 1-year disease-free survival rate. Forest plot of the Odds Ratio(OR) of the 1-year disease free survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g006 Figure 6 3-year disease-free survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. Publication bias Publication bias might exist when nonsignificant findings remain unpublishedthus artificially inflating the apparent magnitude of an effect.The funnel plots of the study are shown in Figure 7.The funnel plots of the 1-year survival rate following VATS and thoracotomy for the treatment of metastatic lung cancer showed asymmetry which suggested that there was some publication bias. .0085329.g007 Figure 7 Funnel plot of the outcome of 1-year survival rate. Discussion Many tumors can metastasize to the lungand colorectal and breast tumors are the most common primary tumors[9].Pulmonary resection has been shown to be beneficial for patients with resectable and isolated pulmonary metastases[10]. Traditional open thoracotomy and VATS are two principally different surgical methods for pulmonary metastasectomy.The selection of an approach depends more on the theoretical knowledge and personal experience of the surgeon than on the evidence. Over the past two decades several studies have demonstrated the benefits of VATS that included less postoperative pain shorter hospital stays a smaller degree of immunosuppression and enhanced recovery and the ability to tolerate adjuvant therapy[11]“[13]. Whether the long-term advantages are comparable to those of open thoracotomy is not well documented. The major deficiency of the VATS approach is that nodules might be undetected by VATS that might be detected by manual palpation during thoracotomy; such missing nodules are not imaged on a preoperative CT scan. The VATS approach has long been controversial because VATS does not consistently detect all the metastases and it is recognized that complete resection remains a major determining factor of survival [14].The detection rate of HRCT for pulmonary metastases is 78“84%[15]“[17].Kayton[18]found that 35% of the pathologically verified metastases were missed by CT. In the International Registry of Lung Metastases study of 5206 patients the 5-year survival rate was 36% for complete resection compared with 13% incomplete resectoin[19]. It is not certain whether the nodule imaged on a CT scan and resected by VATS is the correct one [14]. Those who disagree with the use of VATS hypothesize that VATS-related recurrence is commonly observed including port-site recurrence and resection stump recurrence[20]. Johnstone reported 23 cases of port-site chest wall recurrence related to VATS[21]. They hypothesized that the thoracoscopic approach should only be used in patients with a solitary lesion and when resection is requried for diagnostic purposes. The surgeons who favor the VATS approach advocate that VATS minimizes pain and trauma to the patients and that the VATS group might have an improved tolerance of chemotherapy which would likely ensure delivery of planned post-resection adjuvant therapy without a reduction in dosage or delay. The standard surgical procedure for pulmonary metastases is wedge resection that usually does not require manipulation of the pulmonary hilum which is appropriate for the VATS approach.They hypothesiezd that a lesion overlooked by CT but detected by palpation might not result in a survival gain[22] [23] and may be partially compensated for by carefully follow-up.Flores[24] hypothesized that the VATS group might demonstrate a great number of metachronous tumors over time;however the metachronous lesions in each group was similar. Our work suggests that thoracoscopic resection of metastatic lung cancer is a safe and curative procedure with 13 and 5-year survival rates comparable to those of thoracotomy. Patients with metastatic lung cancer are likely to relapse in the lung and after lung metastasectomy by VATS patients might benefit from a second metastasectomy. We hypothesize that earlier chemotherapy and radiation are essential to maximizing survival. Our study might be subject to pretreatment selection bias because most of the patients selected for open thoracotomy had multiple lesions and high risk and were not suitable for treatment with VATS.The missing lesions perhaps skewed the data more toward VATS as an equivalent procedure. We were also interested in the recurrence of cancerand the disease-free survival rates were evaluated. This study demonstrates a similar 1-year disease-free survival rate;however the 3-year disease-free survival rate is inferior for three reasons. First unrelated cancer deaths were included in our analysis of the 13 and 5-year overall survival which might account for VATS having a comparable overall survival rate but an inferior disease-free survival rate. Secondthe patients in the VATS group might have lesions that are missed and there are more likely to relapse in the lung leading to the inferior 3-year disease-free survival rate.Third some of our included studies were in the early period of VATS development when the technology was immature and some of the complications can now be prevented with more experience. Schaeff[25] reported 23 cases of port-site recurrence associated with VATS that occurred before 1998.The number of cases studied was small and the observation period was limitied. Spiral computed tomography has a far higher detection rate today than it did 20 years ago;so small lesions can be accurately localized before surgery[26] which ensures the success of VATS. With advances in imaging technology palpaiton during open thoracotomy is becoming less important.The latest VATS technology has a high-definition resolution and the flexible-tip thoracoscope enables complete inspection of the pleural cavity.These advancements ensure that VATS is an ideal method for patients with a solitary and relatively small peripheral lesions.Tamas[27] hypothesizes that palpation is necessary in a therapeutic metastasectomy as opposed to a diagnostic procedure.Whether patients with multiple lesions should be treated with open thoracotomy or VATS is controversial. This study is the first meta-analysis of the oncological outcome of thoracoscopic surgery for the treatment of metastatic lung cancer. In our work we observed that VATS might be a promising treatment for metastatic lung cancer. No randomized trials existing to guide doctors in the field of metastatic lung cancer currently. A prospective randomized study of the different surgical strategies is needed. Limitation No randomized controlled trials existing to comparing VATS with thoracotomy have been conducted. Heterogeneity was observed between the sample size and the years covered. Most studies are limited to small observational studies and single-institution case series. For these reasonsthere are only a total of 546 patients were included in the two groups for a study period spans more than a decade. Two of the studies comprise almost 65% of the patients and one study has only 20 patients; there are potential sources of bias in our work.Additional randomized controlled trials in the studies we accessed would have increased the strength of our results.There is a bias for the English language. Conclusion In our meta-analysis we found that for patients with metastatic lung cancer comparing VATS with thoracotomy showed almost equivalent survival rates. The VATS can not replace open thoracotomy completely. Further study is neededand a large multicenter randomized trial comparing VATS and thoracotomy would be ideal. Supporting Information Checklist S1 PRISMA Checklist. (DOC) Click here for additional data file. References 1 RuschVW (2010) Pulmonary metastasectomy: a moving target. J Thorac Oncol5: S130“13120502246 2 CassonAG PutnamJB NatarajanG JohnstonDA MountainC et al (1992) Five-year survival after pulmonary metastasectomy for adult soft tissue sarcoma. Cancer69: 662“6681730117 3 van HalterenHK van GeelAN HartAA ZoetmulderFA (1995) Pulmonary resection for metastases of colorectal origin. Chest107: 1526“15317781341 4 KandiolerD KromerE TuchlerH EndA MullerMR et al (1998) Long-term results after repeated surgical removal of pulmonary metastases. Ann Thorac Surg65: 909“9129564899 5 McCormackPM BainsMS BeggCB BurtME DowneyRJ et al (1996) Role of video-assisted thoracic surgery in the treatment of pulmonary metastases: results of a prospective trial. 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Int J Clin Oncol10: 81“8515864692 10 PorterGA CantorSB WalshGL RuschVW LeungDH et al (2004) Cost-effectiveness of pulmonary resection and systemic chemotherapy in the management of metastatic soft tissue sarcoma: a combined analysis from the University of Texas M. D. Anderson and Memorial Sloan-Kettering Cancer Centers. J Thorac Cardiovasc Surg127: 1366“137215115994 11 PetersenRP PhamD BurfeindWR HanishSI TolozaEM et al (2007) Thoracoscopic lobectomy facilitates the delivery of chemotherapy after resection for lung cancer. Ann Thorac Surg83: 1245“1249 discussion 1250.17383320 12 PaulS AltorkiNK ShengS LeePC HarpoleDH et al (2010) Thoracoscopic lobectomy is associated with lower morbidity than open lobectomy: a propensity-matched analysis from the STS database. 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J Formos Med Assoc106: 911“91818063512 27 MolnarTF GebitekinC TurnaA (2010) What are the considerations in the surgical approach in pulmonary metastasectomy? J Thorac Oncol5: S140“14420502249 28 NakajimaJ TakamotoS TanakaM TakeuchiE MurakawaT et al (2001) Thoracoscopic surgery and conventional open thoracotomy in metastatic lung cancer. Surg Endosc15: 849“85311443456 29 MutsaertsEL ZoetmulderFA MeijerS BaasP HartAA et al (2002) Long term survival of thoracoscopic metastasectomy vs metastasectomy by thoracotomy in patients with a solitary pulmonary lesion. Eur J Surg Oncol28: 864“86812477479 30 NakasA KlimatsidasMN EntwisleJ Martin-UcarAE WallerDA (2009) Video-assisted versus open pulmonary metastasectomy: the surgeon's finger or the radiologist's eye? Eur J Cardiothorac Surg36: 469“47419464921 31 CarballoM MaishMS JaroszewskiDE HolmesCE (2009) Video-assisted thoracic surgery (VATS) as a safe alternative for the resection of pulmonary metastases: a retrospective cohort study. 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Molecular cancer research : MCR 1541-7786 1557-3125 24202705 3946989 10.1158/1541-7786.MCR-13-0300 NIHMS538386 œNEDD9 Depletion Leads to MMP14 Inactivation by TIMP2 and Prevents Invasion and Metastasis. McLaughlin Sarah L. 5 * Ice Ryan J. 5 * Rajulapati Anuradha 5 Kozyulina Polina Y. 1 Livengood Ryan H. 4 Kozyreva Varvara K. 5 Loskutov Yuriy V. 5 Culp Mark V. 3 Weed Scott A. 2 5 Ivanov Alexey V. 1 5 Pugacheva Elena N. 1 5 # 1Department of Biochemistry West Virginia University School of Medicine Morgantown WV 26506 2Department of Neurobiology and Anatomy West Virginia University School of Medicine Morgantown WV 26506 3Department of Statistics West Virginia University School of Medicine Morgantown WV 26506 4Department of Pathology West Virginia University School of Medicine Morgantown WV 26506 5Mary Babb Randolph Cancer Center West Virginia University School of Medicine Morgantown WV 26506 #Corresponding author: Elena N. Pugacheva Mailing address: Department of Biochemistry and Mary Babb Randolph Cancer Center PO Box 9142 1 Medical Center Drive West Virginia University School of Medicine Morgantown WV 26506. Phone: (304) 293-5295; Fax: (304) 293-4667; [email protected] S.L. McLaughlin* and R. J. Ice* contributed equally to this work. 17 12 2013 07 11 2013 1 2014 01 1 2015 12 1 69 81 The scaffolding protein NEDD9 is an established pro-metastatic marker in several cancers. Nevertheless the molecular mechanisms of NEDD9 driven metastasis in cancers remain ill defined. Here using a comprehensive breast cancer (BCa) tissue microarray it was show that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly it was shown that NEDD9 overexpression is a hallmark of highly invasive BCa cells. Moreover NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo leading to decreased circulating tumor cells (CTCs) and lung metastases in xenograft models. Mechanistically NEDD9 localized to invasive pseudopods and was required for local matrix degradation. Depletion of NEDD9 impaired invasion of cancer cells through inactivation of membrane-bound matrix metalloproteinase MMP14 by excess TIMP2 on the cell surface. Inactivation of MMP14 is accompanied by reduced collagenolytic activity of soluble metalloproteinases MMP2 and MMP9. Re-expression of NEDD9 is sufficient to restore the activity of MMP14 and the invasive properties of BCa cells in vitro and in vivo. Collectively these findings uncover critical steps in NEDD9-dependent invasion of BCa cells."
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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 Tai™an Campus of the 960th Hospital of the Chinese People™s Liberation Army No217 Huanshan Road Taishan District Tai™an 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 Dulbecco™s modified Eagle™s 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 real‘time polymerase chain reaction qRT‘PCRThe 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 BeyotimeDual‘luciferase 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 pull‘down 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 Student™s 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 a0HG‘induced 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 a0miR‘‘5pLncRNA 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 a0HG‘induced podocytes injury via a0targeting miR‘‘5pAs 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 d“f 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 miR‘‘5p to a0facilitate PPARÎ expressionAs appeared in Fig a04a“d 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 a0HG‘induced 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 dose“response 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 People™s Hospital Heze Shandong China Department of Nephrology Liaocheng People™s Hospital Liaocheng Shandong China Department of Nephrology Tai™an Campus of the 960th Hospital of the Chinese People™s Liberation Army No217 Huanshan Road Taishan District Tai™an 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 therapies”new 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 Z Wan J Hou X Geng J Li X Bai X MicroRNA27a promotes podocyte injury via PPARgammamediated betacatenin activation in diabetic nephropathy Cell Death Dis 201783e2658 Duan LJ Ding M Hou LJ Cui YT Li CJ Yu DM Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA377 targeting of PPARgamma in diabetic nephropathy Biochem Biophys Res Commun “ Liang W Sun F Identification of pivotal lncRNAs in papillary thyroid cancer using lncRNA“mRNA“miRNA ceRNA network analysis PeerJ 20197e7441 Li D Lu Z Xu Z et al Spironolactone promotes autophagy via inhibiting PI3KAKTmTOR signalling pathway and reduce adhesive capacity damage in podocytes under mechanical stress Biosci Rep 2016364e00355 Susztak K Raff AC Schiffer M Bottinger EP Glucoseinduced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy Diabetes “ Chen Z Ma Y Yang Q et al AKAP1 mediates high glucoseinduced mitochondrial fission through the phosphorylation of Drp1 in podocytes J Cell Physiol https doi101002jcp29646 Jiang L Cui H Ding J Smad3 signalling affects high glucoseinduced podocyte injury via regulation of the cytoskeletal protein transgelin Nephrology https doi101111nep13701 Hu M Wang R Li X Fan M Lin J Zhen J et al LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucoseinduced podocyte injury via its interplay with betacatenin J Cell Mol Med “ Jiao H Xie D Qiao Y LncRNA PRINS is involved in the development of nephropathy in patients with diabetes via interaction with Smad7 Exp Ther Med “ Kim H Dusabimana T Kim SR Je J Jeong K Kang MC et al Supplementation of abelmoschus manihot ameliorates diabetic nephropathy and hepatic steatosis by activating autophagy in mice Nutrients Wu F Li S Zhang N Huang W Li X Wang M et al Hispidulin alleviates highglucoseinduced podocyte injury by regulating protective autophagy Biomed Pharmacother “ Sc
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 stratification 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 fields 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 significance thesefindings 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 profilesof aml specimens and the corresponding clinical followupdata were downloadedidentification 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 identified between high and low score groups stratified bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsignificance 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 identification of gene coexpressionnetworksthrough highthroughput expression profiles 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 networkbriefly 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 mirna“mrna and mirna“lncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a protein“protein interaction ppi networkof the degs identified in the cerna network the interactingpairs with a confidence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplan“meier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical significance 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 kruskal“wallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression profiles 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 classification 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 wasˆ’ range fromˆ’ 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_exehtml6httpwwwtargetscan7http12920671508tringdbidentification of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identified 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 kaplan“meier 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 identified 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 identification 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 final lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aprotein“protein 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 confirmed 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 protein“protein 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 protein“protein 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 mayfind 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 kaplan“meier 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 identified 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 identified 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 specificity 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 pro‚ammatory 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 significance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsignificance 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 identified lncrnas with clinical significance 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 identified 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 marrowmicroenvironment“home 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 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sarscov2 has resulted in numerous cases of coronavirus disease covid19 worldwide in addition to feverand respiratory symptoms digestive symptoms also are observed in some patients with covid19 angiotensinconverting enzyme ace2 was reported to be the receptor for sarscov2 the aim of this study was tocomprehensively investigate the digestive symptoms that occur in covid19 patients and the potential pathogenicroute of the sarscov2 infection in digestive tract ans from the oral cavity to the gastrointestinal tract weinvestigated the digestive symptoms of patients with covid19 and explored ace2 expression in digestive tractand lung cancers based on a series of bulk and singlecell rna sequencing data obtained from public databases wefound that of the patients with covid19 suffered from digestive symptoms among which pharyngalgia was the most common manifestation followed by diarrhea anorexia and nausea the bulktissue rna sequencing analysis indicated that digestive tract ans had higher ace2 expression levels compared tothe lung and the expression of ace2 in the lung increased with age singlecell rnaseq results showed that theace2positivecell ratio in digestive tract ans was significantly higher compared to the lung ace2 expression washigher in tumor cells compared to normal control nc tissues while in gastric tissues ace2 expression graduallyincreased from chronic gastritis to metaplasia to early cancer our data might provide a theoretical basis for screeningthe sarscov2 susceptible population and for the clinical classification of treatment of patients with covid19introductionthe global pandemic coronavirus disease covid19 caused by severe acute respiratory syninfection1 hasdrome coronavirus sarscov2been raging throughout the world by june covid19 has been found in countries with a totalof million confirmed cases worldwide includingover deaths2correspondence yujie liang yujie0350126com uiqing liao drliaoguiqinghotmailcom1department of oral and maxillofacial surgery hospital of stomatology sunyatsen university 56th lingyuanxi road guangzhou guangdongchina2guangdong province key laboratory of stomatology no 2nd zhongshanroad guangzhou guangdong chinafull list of author information is available at the end of the these authors contributed equally jiabin xu mei chu fan zhongedited by i amelioit has been established that sarscov2 invadeshost cells by binding to the transmembrane receptorangiotensinconverting enzyme ace23 which also isthe receptor for sarscov and hcovnl6345 ace2plays a crucial role in the cellular entry for sarscov2which means that ace2positive cells may act as targetcells and are susceptible to infection6 thus the expression of ace2 might affect the invasion path and pathogenicity of the virusthe common symptoms of covid19 include feverfatigue cough myalgia and dyspnea7“ also somepatients may suffer from digestive system symptoms suchas pharyngalgia diarrhea nausea vomiting and abdominalpain10 suggesting that the digestive tract ans also maybe targeted by the virus studies have reported that ace2was expressed in lung esophagus epithelial cells ileum11colon12 kidney bladder13 and oral mucosa14 however 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 httpcreativecommonslicensesby40official of the cell death differentiation association 0cxu cell death discovery page of table demographics and clinical characteristics ofpatients with covid19total number of cases notable ace2 expression in aerodigestive cancerscancersample type samplesize nomeanexpressionlog2 fpkm standarddeviation “lungoralnctumornctumoresophagus ncstomachcolontumornctumornctumorage yearmedianrangeage group no years‰¥ yearssex nomalefemalecomorbidity nohypertensiondiabeteschronic obstructive pulmonary disease copdchronic renal diseasescardiovascular and cerebrovascular diseaseschronic liver diseasesmalignancydigestive system diseasesigns and symptoms nofevercoughdigestive symptomspharyngalgiadiarrheaanorexianauseafatiguedyspneainformation is lacking concerning the comparative analysisof ace2 expression in the entire digestive tract from theoral cavity through the gastrointestinal tract the oropharynx and gastrointestinaltract are physiologicallyinterlinked and share similar microbial environments15they also share similarities with respect to the microenvironment associated with epithelial carcinogenesisreports show that oral bacteria play a role in the genesis ofdigestive tract tumors16“ more than of covid19cases coexist with malignant tumors10 it is unclear whether there are differences in ace2 expression betweentumor and normal cellsin this study we assessed the clinical characteristicsand investigated the digestive symptoms of patientswith covid19 in guangzhou eighth people™s hospitalofficial of the cell death differentiation associationnote data was downloaded from the cancer genome atlas tcgaace2 angiotensinconverting enzyme nc normal control ace2 angiotensinconverting enzyme nc normal controlwe explored ace2 expression in digestive tract cancersand lung cancers based on a series of bulk tissue rnasequencing data from two independent databases andsinglecelltranscriptome data from three singlecelldatabases the results showed that nearly a quarter ofcovid19 patients had gastrointestinal symptoms thatace2 expression was higher in the digestive tract thanthe lung and the ratio of ace2positive cells in tumortissues was higher than that in paracancerous normaltissuematerials and methodspatients™ involvement and data collectionfortyeight hospitalized patients admitted from january to march in guangzhou eighth people™shospital the hospital exclusively designated for covid19patients in guangzhou who were clinically diagnosed withœviral pneumonia based on their clinical symptoms feveror respiratory symptoms with typical changes in chestradiology were preliminarily included in this studypatients without or with negative test results were excludedfrom this studydemographiccharacteristicsincluding medical history comorbidities signs andsymptoms were obtained from the electronic medicalrecord system of guangzhou eighth people™s hospital andanalyzed by three independent researchers all patientsincluded in our study provided consent for the nasopharyngeal swabs and clinicalinformation collectionthis study was approved by the institutional ethics boardof guangzhou eighth people™s hospital no and complied with the declaration of helsinki concerningmedical research using human subjectsinformationclinicalnucleic acid detection of sarscov2nasopharyngeal swabs were collected and placed intoa sterile tube containing rna preservation solution 0cxu cell death discovery page of fig the expression of ace2 in different tissues a“c ace2 expression of different tissues downloaded from tcga database a ace2 expressionin all samples b ace2 expression in tumor samples c ace2 expression in nc samples black solid line median value black dotted line interquartilerange d ace2 expression in cancer cell lines downloaded from ccle database solid line mean value dotted line median value ace2 angiotensinconverting enzyme tcga the cancer genome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinomahnsc head and neck squamous cell carcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinoma cclecancer cell line encyclopediathe swabs were sent for sarscov2 rna extractionand detection within h by a realtime reverse transcriptional polymerase chain reaction rtpcr systemby following the commercial test kit instructions da™angene cooperation cat da0930 as previously described19 briefly two pcr primer and probe sets targetingorf1ab and ncovn genes were separately added intothe same reaction tube positive and negative controlswere involved for detectionbulk tissue rna sequencing rnaseq data analysisthe rnaseq data level and clinical information forsamples were downloaded from the cancer genomeatlas tcgahttpsportalgdccancergovrepositoryusing the gdc data transfer tool the datasets used forace2 analysis included head and neck squamous cellcarcinoma hnsc esophageal carcinoma esca stomach adenocarcinoma stad colon adenocarcinomacoad lung adenocarcinoma luad and lung squamous cell carcinoma lusc gene expression waspresented as log2 fragments per kilobase per millionmapped reads fpkm the effects of age and sex onace2 expression were explored we also downloaded theboxplot for ace2 expression in human cancer cell linesfrom the cancer cell line encyclopedia ccle httpsportalsbroadinstituteccle database for verificationsinglecell rna sequencing scrnaseq data analysisthe lung cancer dataset exp0068 downloaded fromthe cancer singlecell state atlas httpbiocchrbmueducncancerseahomejsp and the lung tissue datasetsra878024 downloaded from the panglaodb httpspanglaodbseindexhtml were merged for further analysis the esophageal squamous cell carcinoma esccdataset gse81812 oral cancer dataset gse103322 gastriccancer dataset gse134520 and colorectal cancer datasetgse81861 were obtained from the gene expressionomnibushttpswwwncbinlmnihgovgeofor gse103322 cells from lymph nodes were removedthe r package seurat version was used for singlegeoofficial of the cell death differentiation association 0cxu cell death discovery page of fig the expression of ace2 in different ans grouped by age a“e the expression of ace2 in different tissues downloaded from tcgadatabase grouped by age top row the expression of ace2 in tumor samples bottom row the expression of ace2 in nc samples bold black linemedian value dotted black line range of values statistical tests mann“whitney utest ace2 angiotensinconverting enzyme tcga the cancergenome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinoma hnsc head and neck squamous cellcarcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinomalogtransformed and scaledcell data analysis genebybarcode count matrices werenormalizedfollowed bydimension reduction using principal components analysispca uniform manifold approximation and projectionumap was used to carry out dimensionality reductionand clusteringall analyses were performed in r r version andgraphpad and the level of significance was set at p ‰¤ resultsdemographics and clinical characteristicsa total of patients diagnosed as covid19 wereincluded in the study twentyfive patients were male and were female the median patient age was years andranged from to years the majority of thepatients included in the study were under years of age of the patients had at least one underlyingcomorbidity the most common of which was hypertension three patients suffered from diabetes chronicobstructive pulmonary disease copd or chronic renaldiseasesrespectively two patients presented withcardiovascular and cerebrovascular disease one patienthad chronic liver disease malignancy or digestive systemdisease respectively the most common symptoms werefever or cough followed by digestive symptoms fatigue or dyspnea among patientswho presented with digestive system symptoms pharyngalgia was the most common manifestationfollowed by diarrhea anorexia and nausea table bulk tissue rnaseq data analysisa total of tumor samples and normal control nc samples were downloaded from the tcgadatabase table shows the expression of ace2log2fpkm in the different samples figure 1a“crespectively show ace2 expression in all tumor andnc samples from the different tissues the resultsshowed that digestive tract ans had higher ace2expression compared to the lung in digestive tractcancers ace2 expression gradually increased from theoral cavity to the esophagus stomach and the colonofficial of the cell death differentiation association 0cxu cell death discovery page of fig the expression of ace2 in different ans grouped by sex a“e the expression of ace2 in different tissues downloaded from tcgadatabase grouped by sex top row the expression of ace2 in tumor samples bottom row the expression of ace2 in nc samples bold black linemedian value dotted black line range of values statistical tests mann“whitney utest ace2 angiotensinconverting enzyme tcga the cancergenome atlas nc healthy normal control luad lung adenocarcinoma lusc lung squamous cell carcinoma hnsc head and neck squamous cellcarcinoma esca esophageal carcinoma stad stomach adenocarcinoma coad colon adenocarcinomafollowing the path of the digestive tract fig1a“c theresults were validated using the cancer cell line datafrom the ccle dataset fig 1dto analyze the effect of age or sex on ace2 expression thepatients were divided into a young group and an oldergroup ‰¥ based on age fig and the patients also wereseparated into females and males fig based on the clinicaldata in the tcga database the results revealed that for lungcancer based on either tumor samples or nc samples fig2a ace2 expression in the older group was higher comparedto the young group also for oral cancer the expression oface2 was significantly increased in the older group for thenc samples fig 2b as well as in the female group for thetumor samples fig 3bscrnaseq data analysissix different scrnaseq datasets were included in thisstudy sra878024 and exp0068 were lung normal tissueor lung cancer scrnaseq data respectively gse103322was oral cancer scrnaseq data gse81812 was scrnaseq data for the escc cell line kyse180 treated withdifferent doses of radiotherapy gse134520 includedscrnaseq data from chronic gastritis cg wild intestinal metaplasia wm severe intestinal metaplasia smand gastric cancer gc gse81861 included scrnaseqdata from colon cancer and adjacent normal tissues theresults showed that ace2positive cells accounted for thelowest proportion in lung samples with inthe nc group and in the tumor groupfig 4a the ace2positive rate in oral cancer cells was fig 4b while the ace2positive ratio inkyse180 treated with different doses of radiotherapywas for gy for gy and for gy fig 4c the ratios of ace2positive cells in the cg wm sm and gc groups were and respectively fig 4d the ace2positivecell ratio was in normal colonicmucosal cells and in colon cancer fig4e the ace2positive rate in the esophageal and oralmucosa was reported to be nearly and respectively we found that the ace2positivecell ratioofficial of the cell death differentiation association 0cxu cell death discovery page of fig see legend on next pageofficial of the cell death differentiation association 0cxu cell death discovery page of see figure on previous pagefig singlecell sequensing analysis of aerodigestive cancer cells a singlecell analysis of lung cancer cells tumor in exp0068 and nc cells insra878024 b singlecell analysis of oral cancer cells in gse103322 c singlecell analysis of escc cell line kyse180 tumor treated with different doses ofradiotherapy in gse81812 d singlecell analysis of cg wm sm and gc cells in gse134520 e singlecell analysis of colon cancer cells tumor and nc cells ingse81861 left column umap plots showing the distribution of cells colorcoded for pathology middle column umap plots showing the distribution oface2positive cell red right column stacked barplot showing the proportion of ace2positive cells red exp0068 was downloaded from the cancer singlecell state atlas sra878024 was downloaded from the panglaodb gse103322 gse81812 gse134520 and gse81861 were obtained from the geneexpression omnibus nc healthy normal control escc esophageal squamous cell carcinoma cg chronic gastritis wm wild intestinal metaplasia sm severeintestinal metaplasia gc gastric cancer umap uniform manifold approximation and projection ace2 angiotensinconverting enzyme study were younger and presented with fewer underlyingdigestive system diseases the most common digestivesymptom was pharyngalgia which was consistent withother studies2224we observed that the expression of ace2 in the lungincreased with age but was independent of sex which wasconsistent with the report by chen 25 this maypartly explain why older patients with covid19 aremore likely to develop pneumoniathis study found that ace2 was highly expressed indigestive tract tumors or paracancerous tissues compared tothe lung for both the bulk tissue analysis and the singlecellanalysis elevated ace2 expression in the digestive tract suggested that the digestive tract ans also should be consideredto be vulnerable targets for sarscov2 infection it wasreported that sarscov2 might cause a cytokine storm andmultian failure in severe pneumonia patients26 similarlysarscov2 might interact with ace2 in digestive anscausing further damage to the mucous membrane barrier andincrease inflammatory cytokine production oralrelatedsymptoms are rarely reported with covid19 infectionsbut this does not indicate that an oral infection route forsarscov2 should be excluded the oral cavity and gastrointestinal ans share similarities in the microbiomeinflammation and tumorigenesis thus we expect that theoral and gastrointestinal ans should exhibit commoninteractions in the route of sarscov2 infection furthermore recent studies have reported a high positive rate ofsarscov2 detection in saliva2728indicating that oralinfection could be an early symptom of covid19interestingly we found that the ace2positive rate intumor cells was significantly higher compared to the ncgroup moreover ace2 expression in gastric tissuesgradually increased from chronic gastritis to intestinalmetaplasia to early gastric cancer similar findings werereported for the colon29 these results suggested thatcancer patients might have a higher risk of sarscov2infection from another perspective we suspected thatincreased expression levels of ace2 affected the occurrence of digestive tract tumors ace2 hydrolyzes ang iito ang1“ which negatively regulates the active reninangiotensin system ras reports show that ang1“plays an inhibitory role in the genesis and development offig ace2 expression increases along with the path of digestivetract ace2positive cell proportion in aerodigestive cancers ace2positive cell proportion in oral mucosa reported by xu 14 ace2positive cell proportion in esophageal epithelium reported by zou 11 ace2 angiotensinconverting enzyme nc healthy normalcontrol cg chronic gastritis wm wild intestinal metaplasia sm severeintestinal metaplasia gc gastric cancerin digestive tract ans was significantly higher than inthe lung the ace2 expression also was higher in tumorcells compared to nc tissues the ace2 expression ingastric tissues gradually increased from chronic gastritisto metaplasia then cancer fig discussionthis study investigated the gastrointestinal symptoms of patients with covid19 who were admitted to theguangzhou eighth people™s hospital we explored ace2expression in digestive tract cancers and lung cancers basedon both bulk tissue rnaseq data and scrnaseq datathe median age of the patients was years whichwas similar to data reported by xu years21but was younger than the ages found in many otherreports892223 consistent with other recent reportshypertension and diabetes were the most commonunderlying comorbidities and fever and cough were themost common symptoms of covid19 infection923 inthis study of the covid19 patients exhibiteddigestive symptoms which was lower than the percentage reported by xu 21 and zhang 22 thereason for this difference may be that the patients in thisofficial of the cell death differentiation association 0cxu cell death discovery page of tumors3031 however sarscov2 could cause aninflammatory cytokine storm when the disease developsinto a longterm chronic state a potential risk mightdevelop for malignant changes to occur in the mucosaunder the effect of various cytokines therefore we suggest that when following patients with covid19 the riskof tumorigenesis should be consideredin summary the ace2positive cells in digestive tract tissuesmight provide possible routes for sarscov2 infectionace2 expression in lung tissue increased with age whichmight explain at least partially why older patients withcovid19 are more likely to develop pneumonia ace2expression was correlated to histological grading suggestingthat cancer patients might be more susceptible to sarscov future research should focus on whether the expression oface2 in digestive tract ans could affect the replication ofsarscov2 and whether sarscov2 infection could affectthe genesis or development of tumorsacknowledgementsthis work was supported by the guangdong financial fund for highcaliberhospital constructionauthor details1department of oral and maxillofacial surgery hospital of stomatology sunyatsen university 56th lingyuanxi road guangzhou guangdongchina 2guangdong province key laboratory of stomatology no 2ndzhongshan road guangzhou guangdong china 3guanghua schoolof stomatology sun yatsen university 56th lingyuanxi road guangzhou guangdong china 4guangzhou eighth people™s hospitalguangzhou medical university guangzhou guangdong china5school of stomatology wuhan university 237th luoyu road wuhanhubei chinaconflict of interestthe authors declare that they have no conflict of interestpublisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreceived may revised july accepted july references coronaviridae study group of the international committee on taxonomy ofviruses the species severe acute respiratory syndromerelated coronavirusclassifying 2019ncov and naming it sarscov2 nat microbiol “ who coronavirus disease covid19 situation report101 httpswwwwhointemergenciesdiseasesnovelcoronavirus2019situationreportswho xu x evolution of the novel coronavirus from the ongoing wuhanoutbreak and modeling of its spike protein for risk of human transmission scichina life sci “ he l expression of elevated levels of proinflammatory cytokines insarscovinfected ace2 cells in sars patients relation to the acute lunginjury and pathogenesis of sars j pathol “ official of the cell death differentiation associationli w the s proteins of human coronavirus nl63 and severe acuterespiratory syndrome coronavirus bind overlapping regions of ace2 virology “ zhou p a pneumonia outbreak associated with a new coronavirus ofprobable bat origin nature “ moein s t smell dysfunction a biomarker for covid19int forumallergy rhinol httpsdoi101002alr22587 mao l neurological manifestations of hospitalized patients with covid in wuhan china a retrospective case series study jama neurol httpsdoi101001jamaneurol20201127 huang c clinical features of patients infected with novel coronavirus in wuhan china lancet “ fang z clinical characteristics of coronavirus pneumonia covid19 an updated systematic review medrxiv httpsdoi zou x singlecell rnaseq data analysis on the receptor ace2 expression reveals the potential risk of different human ans vulnerable to ncov infection front med “ zhang h the digestive system is a potential route of 2019ncovinfection a bioinformatics analysis based on singlecell transcriptomes biorxivhttpsdoi10110120200130927806 lin w singlecell analysis of ace2 expression in human kidneys andbladders reveals a potential route of 2019ncov infection biorxiv httpsdoi10110120200208939892 xu h high expression of ace2 receptor of 2019ncov on the epithelialcells of oral mucosa int j oral sci zhang x the oral and gut microbiomes are perturbed in rheumatoidarthritis and partly normalized after treatment nat med “ whitmore s e lamont r j oral bacteria and cancer plos pathog e1003933 chen x poor oral health is associated with an increased risk of esophageal squamous cell carcinomaa populationbased casecontrol study inchina int j cancer “ maisonneuve p amar s lowenfels a b periodontal disease edentulismand pancreatic cancer a metaanalysis ann oncol “ chen w detectable 2019ncov viral rna in blood is a strong indicatorfor the further clinical severity emerg microbes infect “ stuart t comprehensive integration of singlecell data cell “e21 xu x clinical findings in a group of patients infected with the novel coronavirus sarscov2 outside of wuhan china retrospective caseseries bmj m606 zhang j clinical characteristics of patients infected with sarscov2 in wuhan china allergy httpsdoi101111all142382020 wang c horby p w hayden f g gao g f a novel coronavirus 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"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 œLa 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 MITO“Luc 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·kgˆ’ 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 d™Isola Italy [] at a dose of mg·kgˆ’ ˆ™dayˆ’ 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·kgˆ’ ·dayˆ’ [ ] 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·kgˆ’ Imalgene ip and xylazine mg·kgˆ’ 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 ˆ’ °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 Sant™Andrea 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 withœAntigen 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 field™s 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 œResults 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 manufacturer™s 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 Fisher™s 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 œResults 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 Fisher™s 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 œResults 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 Fisher™s 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 ofKras“driven 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
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"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 hodgkin™s 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 patients“in 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
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laparoscopic surgery for rectal cancer is commonly performed in china however compared with open surgerythe effectiveness of laparoscopic surgery especially the longterm survival has not been sufficiently provedmethods data of eligible patients with nonmetastatic rectal cancer at nanfang hospital of southern medical university andguangdong provincial hospital of chinese medicine between and were retrospectively reviewed longterm survival outcomes and shortterm surgical safety were analysed with propensity score matching between groupsresults of cases collated from two institutes matched pairs were analysed after propensity score matching the estimated blood loss during laparoscopic surgery was significantly less than that during open surgery p¼ and the operativetime and hospital stay were shorter in the laparoscopic group both p the postoperative complications rate was inthe laparoscopic group and in the open group p¼ no significant difference was observed between the laparoscopicgroup and the open group in the 5year overall survival rate vs p¼ 5year relapsefree survival rate vs p¼ or 5year cancerspecific survival rate vs p¼ an elevated carcinoembryonic antigen harvested lymph nodes and perineural invasion were independent prognostic factors affecting overall survival and relapsefreesurvivals our findings suggest that open surgery should still be the priority recommendation but laparoscopic surgery isalso an acceptable treatment for nonmetastatic rectal cancerkey words laparoscopic surgery open surgery propensity score matching rectal cancersubmitted february revised april accepted june vc the authors published by oxford university press and sixth affiliated hospital of sun yatsen universitythis 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 0c kl tan introductioncolorectal cancer is the second most commonly diagnosedcancer and the fifth leading cause of cancerrelated death forboth sexes in china in a multidisciplinary approach thatcombines chemotherapy with radiotherapy for the treatmentof colorectal cancer surgery remains the major approach thefirst successful use of laparoscopy in colorectal surgery waspublished in by jacobs laparoscopic surgery hasbeen performed widely in colon cancer all over the world andseveral randomized“controlled trials have demonstrated thatlaparoscopic surgery for colon cancer is safe and feasible withbetter shortterm outcomes including a decrease in postoperative pain a shorter hospital stay and earlier recoveryand equivalent longterm results compared to open surgery[“] however laparoscopic surgery for rectal cancer is morearduous than that for colon cancer so the early clinical trialsexcluded rectal cancer [“] although a few clinical trialshave shown the advantages of laparoscopic rectalcancer resection compared with open surgery [“] both the acosogz6051 and alacart trials did not support the use of laparoscopic surgery for rectal cancer [ ] it is still controversialwhether laparoscopic surgery is suitable for rectal cancer especially for low rectal cancer therefore we conducted thisretrospective cohort study to compare longterm survival outcomes and shortterm surgical safety between laparoscopicand open surgery for nonmetastatic rectal cancer in thechinese population propensity score matching psm was performed for the study designpatients and methodsstudy designall consecutive eligible patients with rectal cancer were confirmed from the department of general surgery of nanfanghospital of southern medical university and the department ofproctology of guangdong provincial hospital of chinesemedicine between january and december these twocenters were members of the southern chinese laparoscopiccolorectal surgery study group demographic clinical pathologic and imaging features together with the management andoutcomes were carefully reviewed written informed consentwas acquired from patients preceding the surgical proceduresthis study was approved by the ethical committee of nanfanghospital and guangdong provincial hospital of chinesemedicine no ze2019052“study subjectsinclusion criteria were patients with clinical stage i“iii rectalcancer who underwentrectal cancerexclusion criteria were patients with i combined operationsextending to the surrounding an ii multiple cancers iiiemergency operation iv conversion to open surgery or vpatients who received neoadjuvant therapyradical surgery forall included cases were classified into two groups based onthe surgical approach which was either laparoscopic or opensurgery the surgical approach was decided by the individualcolorectal surgeon based on a combined assessment of clinicalendoscopic and imaging featuresdata collectiondata were collected in a prospectively maintained databasefrom clinical report forms the demographic and clinicopathological data included age gender body mass index bmi preoperative carcinoembryonic antigen cealocationoperative time estimated blood loss surgical procedure protective ileostomy tumor grade tumor stage and hospital staypreoperative cea was defined as cea measured closest to theoperation time tumor location was divided into the followingthree sections upper rectum above cm from the anal vergemiddle rectum “ cm from the anal verge and lower rectumbelow cm from the anal verge surgical procedures consistedof three categories low anterior resection abdominoperinealtumorfigure flow diagram of patient disposition 0claparoscopic vs open surgery for rectal cancer table baseline characteristics of the study populationcharacteristictotal cohortmatched cohortlaparoscopic groupn¼ open groupn¼ pvaluelaparoscopic groupn¼ open groupn ¼ pvalueage years mean sdgender n malefemalebmi kgm2 mean sdpreoperative cea n 14 ngml ngmltumor location n upper rectummiddle rectumlower rectumtumor stage n iiiiii sd standard deviation bmi body mass index cea carcinoembryonic antigentable operative and pathological results in matched cohorts variablesurgical procedure n low anterior resectionabdominoperineal resectionhartmann™s procedureprotective ileostomy n operative time min median iqrintraoperative blood loss ml median iqrhospital stay day median iqrtumor grade n wellmoderatepoorothersharvested lymph nodes n 15lymphovascular invasion n perineural invasion n tumor deposits n postoperative complications n wound infectionileusurinary dysfunctionanastomosis leakageintraabdominal bleedingpneumoniacardiac eventreoperation n mortality n iqr interquartile rangelaparoscopic group n¼ open group n ¼ “ “ “ “ “ “ pvalueresection and hartmann™s procedure tumor grade was dividedinto three types well differentiated moderately differentiatedand poorly differentiated including signet or mucinous adenocarcinoma tumor stage was based on the final pathologic report and preoperative imaging examinationoutcome measurementsthe primary endpoint of this study was overall survival osrelapsefree survival rfs and cancerspecific survival cssos was defined as the time from operation to death from any 0c kl tan figure survival curve after laparoscopic surgery vs open surgery in matchedcohortscause or the last followup rfs was defined as the time fromoperation to identified recurrence or any cause of death csswas defined as the time from operation to death due to rectalcancer the last followup was january ileus urinary dysfunctionthe secondary endpoints were operative time estimated bloodloss hospital stay reoperation postoperative complications andmortality postoperative complications were defined as woundinfectionleakageintraabdominal bleeding pneumonia and cardiac eventsintraabdominal bleeding was defined in this study as bleeding requiring transfusion or reoperation all complications within daysafter surgery were recorded postoperative mortality was traditionally defined as any death occurring within days after surgeryanastomoticstatistical analysisdata are presented as mean standard deviation or medianwith interquartile range iqr for quantitative variables withparanormal distribution and numbers with percentages for categorical variables quantitative variables were compared usingthe student™s ttest or mann“whitney u test categorical variables were analysed using the chisquare test or fisher™s exacttest the estimates of the differences in age gender bmi preoperative cea level tumor location and tumor stage between thetwo groups were performed using psm [ ]survival rates were calculated by using the kaplan“meiermethod and comparisons between groups were performed withthe logrank test to identify the prognostic factors univariateand multivariate analyses were performed using the cox proportional hazards regression model and the results were presented as hazard ratios hrs with confidence intervalscis only factors with p in the univariate analysis wereevaluated in subsequent multivariate analysis using forwardstepwise selection for os and rfs a p was regarded asstatistically significant all statistical analyses were carried outwith ibmvr spssvr statistics version resultsbaseline characteristicsbetween january and december eligiblepatients were collected from hospitals in china of patients cases were excluded among the remaining cases underwent laparoscopic surgery and underwent open surgery after psm of pairs ofpatients were successfully matched figure baseline characteristics are outlined in table before psm there were differences in age and preoperative cea between the two groups afterpsm all variables were well balancedshortterm surgical outcomesthe perioperative and pathological results in matched cohortsare presented in table the estimated blood loss during laparoscopic surgery was significantly less than that during opensurgery p¼ in the laparoscopic group the operative timeand hospital stay were shorter than in the open groupp the incidence of postoperative complications was in the laparoscopic group and in the open groupp¼ in the open group the most common complicationswere wound infection and pneumonia followed byanastomosis leakage whereas in the laparoscopic groupthe most common complication was anastomosis leakage followed by pneumonia longterm survival outcomesin the matched cohorts the median followup period was months in the laparoscopic group iqr “ monthsand months in the open group iqr “ monthsduring the followup patients died among whom diedfrom rectal cancer and had locoregional recurrence or distantmetastasis no significant difference was observed between thelaparoscopic group and the open group in 5year os vs p¼ 5year rfs vs p¼ or 5yearcss vs p¼ figure subgroup analyses for os were conducted for gender agebmi tumor location and tumor stage compared with open surgery male patients or those with an intermediate bmi to who underwent laparoscopic surgery tended to show worseos figure 0claparoscopic vs open surgery for rectal cancer figure subgroup analysis of overall survival in matched cohortstable multivariate analysis for os and rfs in matched cohortsvariableosrfspreoperative cea vs 14 ngmlnumber of harvested lymph nodes 15 vs perineural invasion yes vs nohr cipvalue“““hr cipvalue“““os overall survival rfs relapsefree survival cea carcinoembryonic antigen hr hazard ratio ci confidence intervalprognostic factors for longterm survivalprognostic factors affecting survival are presented in table univariate analyses revealed that an elevated cea ngml harvested lymph nodes perineural invasion and tumordeposits were associated with poor os and that an elevatedcea harvested lymph nodes perineural invasion and lymphovascular invasion were associated with poor rfs data notshown the surgical approach laparoscopic vs open was notassociated with os hr ci “ and rfs hr ci “ multivariate analyses testified that an elevated cea harvested lymph nodes and perineural invasionwere independent factors affecting os and rfs table discussionlaparoscopic surgery for rectal cancer is commonly performedin many countries nevertheless the evidence for laparoscopicsurgery for rectal cancer is insufficient this study focused onthe longterm survival outcomes and surgical safety of patientswho underwentlaparoscopic or open surgery for nonmetastatic rectal cancer in the chinese population in this twocenter study psm was performed to make selection balance between patients treated with laparoscopic and open surgery thesix factors of age gender bmi preoperative cea level tumor location and tumor stage were used as described in the protocolthe baseline characteristics were ideally balanced between thelaparoscopic and open groupssome studies have reported similar postoperative complications and mortality between laparoscopic surgery and opensurgery for rectal cancer [ ] and other studies have reportedfewer postoperative complications after laparoscopic surgerythan after open surgery [ ] in our study there were no significant differences in postoperative complications includingwound infectionileus urinarytract infection anastomosisleakageintraabdominal bleeding pneumonia and cardiacevent between the two groups the longer operative time is often considered a disadvantage of laparoscopic surgery according to some previous reports [ ] in contrast our studyshowed that the operative time of laparoscopic surgery wasshorter than that of open surgery the clasicc trial and colorii trial both showed that hospital stay was significantly shorterin the laparoscopic group [ ] similarly our study alsoshowed that the hospital stay for laparoscopic surgery wasshorter than for open surgerywith regard to longterm survival no largescale clinical trials have demonstrated a statistically significant difference between laparoscopic and open surgery for rectal cancer thecolor ii trial indicated no statistically significant differences indfs and os between laparoscopic and open surgeries inthe corean study dfs in laparoscopic surgery is noninferiorcompared to that in open surgery for mid or low rectal cancer consistently with previous studies os rfs and css didnot differ in both groups in our study interestingly subgroupanalyses for os showed that male and intermediate bmi to kgm2 subgroups were associated with unfavorable outcomes in the laparoscopicsurgery group vs the opensurgerygroup chinese male populations have a narrow pelvis whichmight affect the visualization of and access to the deep pelvic 0c kl tan anatomy during laparoscopic surgery kitano foundthat laparoscopic surgery might affect longterm outcomes inthe highbmi kgm2 subgroup in the current study thebmi subgroup unfavorable for laparoscopic surgery that weidentified was intermediate bmi not high bmi it might be dueto lower bmi in the chinese population compared to that in thewestern population and the small proportion of patientswith high bmi in our cohort further evaluation will be neededto determine which subgroups of patients require additional attention when undergoing laparoscopic surgerylymphovascularwe evaluated several possible prognostic factors that mayinfluence survival in patients with rectal cancer including tumor location tumor stage tumor grade surgical approach preoperative cea levelinvasion perineuralinvasion and tumor deposits [“] as expected our studyshowed that perineural invasion was the significant prognosticfactor affecting os and rfs perineural invasion refers to the invasion of cancer cells into any of the layers of the nerve sheatha higher grade of perineural invasion was related to local recurrence and metastasis in distant ans such as the liver lungand peritoneum all patients in this study underwent radical surgery with lymphnode dissection a minimum of harvested lymph nodes is recommended to ensure adequatestaging and oncologic resection for colorectal cancer themore lymph nodes harvested the better the prognosis [ ]in this study the average number of harvested lymph nodeswas we found that patients with 15 harvested lymphnodes had better os and rfs than those with harvestedlymph nodes several studies have shown that elevated preoperative cea was a poor prognostic factor in colorectal cancer[“] in our study we also found that patients with an elevated preoperative cea had poorer os and rfsour study has several limitations first a selection biasexisted due to its retrospective design to reduce this the twogroups were matched carefully using psm second the statistical power is insufficient because the number of patients enrolled may not be sufficient after matching third data aboutadjuvant therapy after surgery were not collected which mightbe different between both groups and thus have influenced survival outcomes fourth the exclusion of converted cases mayintroduce a bias in favor of laparoscopic surgery finally thebowelrecovery data could not be exactly assessed due to thelack of records in this retrospective study therefore further research with a large population is still awaitedanydifferencesinin our study revealed the benefit of laparoscopicsurgery on shortterm outcomes including less blood lossshorter operative time and shorter hospital stay we did notfindcomplicationslaparoscopic surgery was similar to open surgery in terms ofos rfs and css for patients however male patients and thosewith an intermediate bmi in the laparoscopic group tended toshow worse os than those in the open group findings fromthis study suggest that open surgery should still be the priorityrecommendation but laparoscopic surgery is also an acceptabletreatment for nonmetastatic rectal cancer in the chinesepopulationpostoperativeauthors™ contributionsklt hjd zqc and tym collected and analysed the dataklt hl and rsx performed statistical analysis klt andhjd drafted the manuscript gxl and xhf performed theprocedure conceived of and designed the study and criticallyrevised all the intellectual content of the manuscript allauthors read and approved the final manuscriptfundingthis work was supported by clinical research of guangdongprovincial hospital of chinese medicine [no yn10101902]and a scientific research project of guangdong provincialacademy of chinese medical sciences [no yn2018ml11]acknowledgementsthe authors thank the patients and their families for making this retrospective study possible we also thank all theinvestigators and staff who contributed to the patientfollowup and data collection in nanfang hospital ofsouthern medical university and guangdong provincialhospital of chinese medicineconflicts of interestthe authors declare that there is no conflict of interests inthis studyreferences feng rm zong yn cao sm current cancer situation inchina good or bad news from the global cancerstatistics cancer commun “jacobs m verdeja jc goldstein hs minimally invasive colonresection laparoscopic colectomy surg laparosc endosc “ hewett pj allardyce ra bagshaw pf shortterm outcomes of the australasian randomized clinical study comparing laparoscopic and conventional open surgical treatmentsfor colon cancer the alccas trial ann surg “ buunen m veldkamp r hop wc survival after laparoscopic surgery versus open surgery for colon cancer longterm outcome of a randomised clinical trial lancet oncol “ clinical outcomes of surgical therapy study group a comparison of laparoscopically assisted and open colectomy forcolon cancer n engl j med “ bonjer hj haglind e jeekel i laparoscopic surgery versusopen surgery for colon cancer shortterm outcomes of arandomised trial lancet oncol “ fleshman j sargent dj green e laparoscopic colectomyfor cancer is not inferior to open surgery based on 5year datafrom the cost study group trial ann surg “ van der pas mh haglind e cuesta ma laparoscopic versus open surgery for rectal cancer color ii shortterm outcomes of a randomised phase trial lancet oncol “ kang sb park jw jeong sy open versus laparoscopicsurgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy corean trial shortterm outcomes of anopenlabel randomised controlled trial lancet oncol “ zhou zx zhao ly lin t longterm oncologic outcomesof laparoscopic vs open surgery for stages ii and iii rectal 0ccancer a retrospective cohort study world j gastroenterol“iii colon cancer jcog0404 a phase randomised controlledtrial lancet gastroenterol hepatol “laparoscopic vs open surgery for rectal cancer fleshman j branda m sargent dj effect of laparoscopicassisted resection vs open resection of stage ii or iii rectalcancer on pathologic outcomes the acosog z6051 randomized clinical trial jama “ stevenson ar solomon mj lumley jw effect oflaparoscopicassisted resection vs open resection on pathological outcomes in rectal cancer the alacart randomizedclinical trial jama “ austin pc an introduction to propensity score methods forreducing the effects of confounding in observational studiesmultivariate behav res “ wang h chen x liu h laparoscopyassisted colectomyas an oncologically safe alternative for patients with stage t4colon cancer a propensitymatched cohort study bmc cancer bonjer hj deijen cl abis ga a randomized trial of laparoscopic versus open surgery for rectal cancer n engl j med“ lujan j valero g biondo s laparoscopic versus opensurgery for rectal cancer results of a prospective multicentreanalysis of patients surg endosc “ landi f vallribera f rivera jp morbidity after laparoscopic and open rectal cancer surgery a comparative analysisof morbidity in octogenarians and younger patientscolorectal dis “ toda s kuroyanagi h laparoscopic surgery for rectal cancercurrent status and future perspective asian j endosc surg“ guillou pj quirke p thorpe h shortterm endpoints ofconventionalinpatients with colorectal cancer mrc clasicc trial multicentre randomised controlled trial lancet “laparoscopicassisted surgeryversus jeong sy park jw nam bh open versus laparoscopicsurgery for midrectal or lowrectal cancer after neoadjuvantchemoradiotherapy corean trial survival outcomes of anopenlabel noninferiorityrandomised controlled triallancet oncol “ kitano s inomata m mizusawa j survival outcomes following laparoscopic versus open d3 dissection for stage ii or mehrkhani f nasiri s donboli k prognostic factors insurvival of colorectal cancer patients after surgery colorectaldis “ wiratkapun s kraemer m seowchoen f high preoperative serum carcinoembryonic antigen predicts metastatic recurrence in potentially curative colonic cancer results of afiveyear study dis colon rectum “ huang qs qin hb xiao j association of tumor differentiation and prognosis in patients with rectal cancer undergoingneoadjuvant chemoradiation therapy gastroenterol rep “ liebig c ayala g wilks ja perineural invasion is an independent predictor of outcome in colorectal cancer j clin oncol“ ueno h shirouzu k eishi y study group for perineuralinvasion projected by the japanese society for cancer of thecolon and rectum jsccr characterization of perineural invasion as a component of colorectal cancer staging am j surgpathol “ amin mb edge sb greene fl eds ajcc cancer stagingmanual 8th edn new york springer “ rosenberg r engel j bruns c the prognostic value oflymph node ratio in a populationbased collective of colorectal cancer patients ann surg “ sjo oh merok ma svindland a prognostic impact oflymph node harvest and lymph node ratio in patients withcolon cancer dis colon rectum “ tarantino i warschkow r worni m elevated preoperative cea is associated with worse survival in stage iiii rectalcancer patients br j cancer “ huang sh tsai ws you jf preoperative carcinoembryonic antigen as a poor prognostic factor in stage iiii colorectal cancer after curativeintent resection a propensity scorematching analysis ann surg oncol “ konishi t shimada y hsu m association of preoperativeand postoperative serum carcinoembryonic antigen and colon cancer outcome jama oncol “ 0c'
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purpose pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer lapc prevents surgical resection this study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationmethods in this phase iii trial patients with lapc were randomised to gemcitabinenab paclitaxel plus arm a n24 or minus arm b n13 pamrevlumab those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria resection rates progression free and overall survival were evaluatedresults eighteen patients in arm a and seven in arm b completed six cycles of therapy with similar toxicity patterns in arms a and b carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively sixteen per cent of patients were radiographically downstaged by national comprehensive cancer network criteria in arm a and in arm b positron emission tomography normalised in vs of patients in arm a vs arm b respectively and correlated with surgical exploration eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in arm a vs arm b p01193 respectively postoperative complication rates were not different between armss neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with lapc without added toxicity this combination merits evaluation in a larger patient cohortintroductionpancreatic cancer is currently the third leading cause of cancer death in the usa1 and by it will likely become the second leading cause of cancer related death after key questionswhat is already known about this subject –º pamrevlumab is anti ctgf1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseasewhat does this study add –º this study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerhow might this impact clinical practice –º this study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged os curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 however only of patients have disease amenable to upfront curative resection at the time of diagnosis4 approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per national comprehensive cancer network nccn guidelines6 patients with locally advanced pancreatic cancer lapc have a prognosis similar to those with metastatic disease with a historical median overall survival os of picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access“ months with recent trials demonstrating median os of months7 recent single institution retrospective studies have reported the potential for resection of lapc with neoadjuvant therapy irrespective of imaging findings with promising results8 however these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation current prospective trials have documented resection rates of lapc in the range of to therefore novel approaches are needed to improve patient outcomesthe tumour biology inherent to pancreatic ductal adenocarcinoma pdac significantly contributes to the poor outcomes seen in this disease notably pdac exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor ctgf expression12 ctgf appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition this leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 this stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticspamrevlumab is a human monoclonal antibody that targets ctgf preclinical studies showed that ctgf overexpression is associated with both desmoplasia and gemcitabine resistance in the kpc pancreatic cancer mouse model14 when pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of xiap an antiapoptotic protein15 when tested in patients with advanced pancreatic cancer stage iv and locally advanced stage iii treated with gemcitabine and erlotinib in a phase iii study n75 pamrevlumab displayed multiple favourable outcomes16we hypothesised that through inhibition of the downstream effects of ctgf overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of pdac tumours with this in mind this novel phase iii randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in lapc with special emphasis on surgical eligibility and safetymethodsstudy designthis was a phase iii randomised trial of safety and efficacy in patients with lapc who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy the randomisation was preplanned and blinded to the investigator the study was approved by individual institutional review boards at nine us institutions and conducted according to the declaration of helsinki the trial was registered at clinicaltrials gov as nct eligibilitykey protocol eligibility requirements included biopsy proven diagnosis of pdac radiographic staging consistent with locally advanced unresectable disease as defined nccn guidelines v2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per response evaluation criteria in solid tumors recist v11 eastern cooperative oncology group ecog performance status of or adequate haematological renal and hepatic function no prior therapy for pdac and no concomitant cancer diagnosis within the past yearsstudy schemaeligible patients were randomised to arm a or arm b to receive a total of six treatment cycles “ weeks of therapy figure patients in arm a received pamrevlumab mgkg by intravenous infusion on days and of each day cycle with an additional dose given on day in the first cycle patients in both arms a and b received gemcitabine mgm2 by intravenous infusion on days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on days and of each day cycle doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care soc15 patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event ae or toxicity withdrew consent or were withdrawn at the investigator™s discretion all patients were followed for drug toxicity until days after the last drug dose patients undergoing surgery were followed for days following hospital discharge for surgical complications ctgf levels were obtained prior to treatment from all patients plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug after all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy postoperative complications including day readmissions and day mortality were notedresponse assessmentpatients were evaluated for response by the following measures carbohydrate antigen ca “ measured at baseline first day of each cycle and end of treatment eot recist v11 read based on full body ct imaging high resolution dual phase fine cut ct imaging at baseline and every weeks thereafter fluorodeoxyglucose fdg positron emission tomography pet imaging and nccn v2 resectability criteria at baseline and eotpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessfigure patient flow and surgery outcomes in arm a four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery in arm a four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive sma encasement in arm b one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement sma superior mesenteric arterysurgical assessmentsubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol pp defined criteria given that patients included in the trial were determined to be initially unresectable by radiographic imaging and nccn criteria objective criteria were developed to standardise attempts at surgical resectionpatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma ca “ level by ‰¥ at eot compared with baseline reduction in fdg pet maximum standardised uptake value suvmax by ‰¥ at eot compared with baseline radiological tumour response per recist of partial response pr or complete response cr at eot or met the definition of resectable or borderline resectable per nccn guidelines subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on ct scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein pvsplenic vein thrombosis pancreatitis or decline in performance status to a karnofsky score ‰¤ or picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes the final decision regarding whether resection was to be performed was made by the treating surgeonendpointssafety endpoints included serious adverse events sae during neoadjuvant therapy and surgical complications postresection the efficacy endpoints included surgical eligibility r0 resection r0r1 resection median os progression free survival pfs and year survival rate all patients were followed and data analysis was stratified by pp population and intention to treat itt cohortstatistical considerationsthe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test exact cis for the point estimates as well as the treatment difference were obtained from the sas proc freq procedure with the exact option the two treatment arms were compared using the cochran mantel haentzel test controlling for baseline factors tnm stage ecog ca “ pet suvmax 0copen accesssuperior mesenteric artery sma involvement coeliac abutment and so on as prespecified in the protocol all cause mortality was used in determining os which was analysed by the kaplan meier method survival status was updated within month before the data cut off date data from patients who were alive at the cut off date were censored for survival analysis all statistical tests were performed at the significance level of α005 using two sided testsresultspatient characteristics and dispositionthirty seven patients were randomised to study treatment to arm a pamrevlumabgemcitabinenab paclitaxel and to arm b gemcitabinenab paclitaxel alone patient characteristics at baseline are summarised in table all patients enrolled were unresectable by nccn criteria patients had tumour arterial involvement sma encasement ° coeliac abutment table patient characteristicsbaseline demographics “ years “ years ‰¥ years median male femaleage group sex bmi kgm2 mean sd median min maxecog grade grade tnm stage t3 n0 m0 t3 n1 m0 t4 n0 m0 t4 n1 m0 t4 nx m0location of the tumour in the pancreas non resectability per nccn criterion head body tail median tumour size mm ° sma encasement any coeliac abutment inferior vena cava invasion or encasement unreconstructible smvportal occlusion aortic invasion and encasementarm agnppn24 arm bgnpn13 totaln37 to to to · ok as isnot mutually exclusivebmi body mass index ecog eastern cooperative oncology group g gemcitabine n number of subjects nccn national comprehensive cancer network np nab paclitaxel p pamrevlumab pv portal vein sma superior mesenteric artery smv superior mesenteric veinpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible pvsuperior mesenteric vein smv occlusion a higher percentage of patients with sma encasement ° were randomised to arm a vs arm b patient disposition is summarised in figure twenty four patients in arm a received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles six patients discontinued treatment early due to progressive disease three patients aes two patients or physician decision one patient thirteen patients in arm b received gemcitabinenab paclitaxel patients completed six treatment cycles six patients discontinued treatment early due to progressive disease two patients aes two patients or patientphysician decision two patientssafetysaes are summarised in table forty one per cent of patients had a treatment emergent sae arm a arm b no individual toxicity category occurred with frequency except systemic infection patients there was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapytable summary of treatment emergent serious adverse eventssystem organ classpreferred term ascites nausea pancreatitis vomiting device occlusion drug withdrawal syndrome feverno of patients with any treatment emergent saeblood and lymphatic disorders haemolytic uremic syndrome lymphadenopathycardiac disorders cardiac failure supraventricular tachycardiagastrointestinal disorders general disorders and administrative site conditions hepatobiliary disorders infections sepsis cellulitis urinary tract infectioninjury poisoning and procedural complications respiratory thoracic and mediastinal disorders skin and subcutaneous disorders cholangitis hyperbilirubinaemia craniocerebral injury pneumonitis pulmonary embolism rasharm an24n arm bn13n overalln37n · ok as ispicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessresponse to therapyin arm a had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria during the treatment period the median ca “ decline was patients were non secretors seven out of patients had best objective recist response crpr some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of ca “ patients or normalisation pet suvmax in in arm b had ‰¥ ca “ decline at eot response by recist pr ‰¥ decline in pet suvmax and were radiographically downstaged by nccn criteria four out of patients had best objective recist response cr pr in arm b of patients had an œexceptional ca “ response and had an ˜exceptional™ pet response as defined by either ‰¥ normalized ca response normalized suv max andorradiographic downstaging post therapy completion surgical evaluationoverall of the total study patients were eligible for surgical exploration using protocol defined criteria arm a arm b p00019 resection was completed in of the patients arm a arm b p01193 details of the nine resected patients are shown in table in arm a of the patients were eligible for surgical exploration in the itt population and of the patients were eligible in the pp population patients who completed six cycles of treatment in arm a out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined eight out of patients in arm a were resected r0 r1 the remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively in arm b of the patients were eligible for surgical exploration in the itt population and were eligible in the pp population of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionpredictors of resectionhigh ca “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 normalisation versus non normalisation of pet suvmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection all nine successful resections were identified by one or both of these criteria table summary of resected patientssitesubject idtreatmentarmresponse to treatmentnccnbaselinenccnend of treatmentresection status“““““““““aaaaaaaab unresectablecoeliacunresectablesma smvunresectablecoeliacunresectablecoeliacunresectablesmvunresectablesmaunresectablesma smv coeliacunresectablesmaunresectablecoeliacunresectablecoeliacunresectablesma smvunresectablecoeliacborderline resectableunresectablesmvunresectablesmaunresectablecoeliacunresectablesmaunresectablecoeliacr0r1r0r0r1r1r1r0r0protocol defined criteria ca “ decrease fdg pet suvmax decrease ‰¥ recist v11 response pr or cr nccn resectable or borderline resectable criteriaca carbohydrate antigen cr complete response fdg fluorodeoxyglucose nccn national comprehensive cancer network pet positron emission tomography pr partial response recist response evaluation criteria in solid tumors sma superior mesenteric artery smv superior mesenteric vein suvmax maximum standardised uptake valuepicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cconversely radiographic features of response did not correlate with operative potential neither recist response nor radiographic downstaging per nccn criteria statistically correlated with completed resectionsurgical complicationspostoperative complications were summarised according to the clavien dindo classification posthoc analysis ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in arm a grade ii there was one episode of clinically significant pancreatic leak in each arm grade iiia no reoperations and no day or day surgical mortality were noted one patient in arm b had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively grade iiib no wound complications or superficial site infections were noted in either group four out of patients and out of patients in arm a and b respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalas of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months pfs was months ci to and months ci to in arm a and arm b respectively one year survival and median os were and months ci open accessto in arm a and and months ci nr in arm b the median os for all patients who were eligible for surgical exploration arm a arm b vs ineligible arm a arm b was months ci nr vs months ci to p00766 the median os for resected arm a arm b vs non resected patients arm a arm b was not reached ci nr vs months ci to p00141 figure discussionthe treatment of lapc with neoadjuvant therapy remains challenging and there is no established soc several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 the combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates a meta analysis of studies using folfirinox has demonstrated resection rates ranging from to in lapc17 one of the larger studies including patients with lapc reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen folfirinox or gemcitabine based18 recently a single institution and single arm prospective study of neoadjuvant folfirinox and losartan with selective use of radiation in patients with lapc reported an r0 resection rate of figure overall survival resected vs non resected patientspicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access however the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease these retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of lapcthe anti ctgf mechanism of action with respect to gemcitabine based therapy a recent large scale prospective trial of patients with lapc treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation more recently the la pact trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with lapc were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 last although folfirinox has been the most studied induction combination chemotherapy regimen in this population recent randomised data from european patients who received neoadjuvant folfirinox versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to r0r1 to resection rate vs p0135 or os vs months p0268given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 this stromal depletion also translated into a decrease of suv uptake on pet22 in the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsthe protocol specified therapeutic response criteria ca “ pet suvmax recist and nccn criteria were used as criteria to determine eligibility for surgical exploration in lapc this is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards nccn criteria for example by nccn conversion alone ie converted from unresectable to borderline resectable only of patients in arm a would have been eligible for surgical exploration however by protocol criteria of patients in arm a were eligible for surgical exploration a higher percentage of patients were eligible for surgical exploration by the above criteria in arm a vs arm b vs respectivelyoverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size of the nine subjects that were successfully resected in this trial only one was converted by nccn criteria to borderline resectable prior to surgical exploration despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti ctgf mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients this hypothesis needs to be confirmed and patients should be stratified by coeliac andor sma involvementthe most common predictive factors for eligibility for surgical exploration and resection were ca “ decline and pet suv max response which are indicators of tumour response to treatment the combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success both the ability of ca “ response and the inability of radiographic response recist and nccn criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials in the mpact study both ca “ and pet response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 recent surgical series of patients with borderline resectable and lapc have also corroborated their impact in the localised setting25 correlation of clinical response with plasma levels of endogenous ctgf and pamrevlumab exposure as shown in the prior study by picozzi et al16 may provide added prognostic and predictive insightwith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel in addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone pamrevlumab is well tolerated and considered safe compared with the soc drugs for patients with pdac these observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in lapc in addition there were no signals of increased surgical morbidity or wound healing problems with ctgf blockade by pamrevlumab in fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationfinally while survival data are not yet mature both patients who were eligible for surgery and those that picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cwere ultimately resected had longer pfs and os highlighting the importance of surgical resection of the tumour therefore more investigation into newer agents targeting lapc and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseasein this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in lapc with prespecified criteria for surgical exploration the use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates further evaluation of this drug combination in the neoadjuvant treatment setting for lapc is warranted and a larger phase iii trial with resection and survival endpoints is ongoingcontributors fibrogen inc was the study sponsor that designed the study in consultation with the principal investigator vp and surgical co investigator fgr all authors except those of the sponsor contributed patients to the study fibrogen was responsible for data collection and analysis all authors reviewed the manuscript and signed off on its accuracyfunding the study was funded by fibrogen inc san francisco cadisclaimer the corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the bmj publishing group ltd and its licensees to permit this article if accepted to be published in esmo open editions and any other bmjpgl products to exploit all subsidiary rights as set out in our licencecompeting interests mc mz sp ek and ec are employees of fibrogen and hold stock andor stock options in fibrogenpatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available on reasonable request all data relevant to the study are included in the article or uploaded as supplementary information data will be available as presented in this manuscriptopen access this is an open access article 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 any changes made are indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idewa a0carrier http orcid org references american cancer society cancer facts figures available httpswww cancer org content dam cancer org research cancer facts and statistics annual cancer facts and figures cancer facts and figures pdf rahib l smith bd aizenberg r et a0al projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the united states cancer r
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The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical managementDehong Che1 Zhirong Yang2 Hong Wei3 Xuedong Wang4 Jiayin GaoID1 Department of Obstetrics and Gynecology The Second Affiliated Hospital of Harbin Medical UniversityHarbin China Department of Ultrasound Harbin Red Cross Center Hospital Harbin China Departmentof Ultrasound The Second Affiliated Hospital of Harbin Medical University Harbin China Department ofObstetrics and Gynecology Longnan Hospital Daqing General Hospital Daqing Chinaa1111111111a1111111111a1111111111a1111111111a1111111111 dsp082901163comAbstract ACCESSCitation Che D Yang Z Wei H Wang X Gao J The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical management PLoSONE e0236725 101371journalpone0236725Editor Linus Chuang University of Vermont LarnerCollege of Medicine UNITED STATESReceived February Accepted July Published August Copyright Che This is an access distributed under the terms of the CreativeCommons Attribution License which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existAbbreviations FIGO International Federation ofObstetrics and Gynecology TVCDFI transvaginalObjectiveTo analyze the relationship of Adler grade by transvaginal color Doppler flow imaging TVCDFI and the clinical pathological parameters of patients with cervical cancer and to identify the value of Adler grade in the diagnosis and treatment of cervical cancerMethodsPatients with cervical cancer diagnosed pathologically in our hospital from January to December were included All patients underwent TVCDFI examination and theimages were divided into to III grades according to the Adler grades and the correlationsbetween the Adler classification and clinical pathological parameters clinical stage masssize pathological type squamous cell carcinoma subtype CA125 CA199 were analyzedResultsA total of patients with cervical cancer were included With the increase of Adler severity the clinical stage of cervical cancer increased accordingly the cancer size differed significantly in patients with different Adler grade p There were significant differences inthe level of CA125 CA199 between the squamous cell carcinoma and adenocarcinoma allp005 the Adler grade was positively related with the clinical stage pathological type andsquamous cell carcinoma subtypes of cervical cancer all p005 no correlations werefound among the Adler grade and the cancer size CA125 CA199 all p005 The areaunder ROC curve of the cervical squamous cell carcinoma predicted by Adler grade basedon FIGO results and pathological results was 0811and respectively all p005ConclusionsAdler grades are closely associated with the clinical pathology of cervical cancer which maybe a convenient and effective approach for the assisting assessment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0ccolor Doppler ultrasound WHO World HealthanizationUltrasound cervical cancerIntroductionCervical cancer is one of the most common malignant tumors in gynecology it has beenreported that the morbidity and mortality of cervical cancer ranks second among gynecological malignancies second only to breast cancer [ ] The number of new cases of cervical cancer has been increased every year around the world and the onset age of cervical cancer hasbecome much younger [] Cervical cancer is a malignant tumor which occurs at the junctionof squamous epithelial cells in the cervical vagina or the transitional zone and columnar epithelial cells in the endometrium of the cervical canal [] The etiology is not totally clear and itmay be related to sexual behavior frequency of deliveries and the infection of human papillomavirus [ ] The early detection and treatment of cervical cancer is very essential to theprognosis of patients with cervical cancerIn recent years with the popularization of many effective screening methods the overallmortality rate of cervical cancer patients has declined [] The clinical diagnosis mainlydepends on clinical manifestations and gynecological examinations which requires the combination of multiple auxiliary diagnostic methods including cervical cytology film cervicalmultipoint biopsy The International Federation of Obstetrics and Gynecology FIGO hasdeveloped a clinical staging standard for cervical cancer which referred to the colposcopypathological biopsy [] However when comparing the early diagnosed stage with the patient™spostoperative medical examination results the stage of cervical cancer has been much underestimated [“] More sophisticated radiological methods such as ultrasound computertomography and magnetic resonance imaging hasn™t been included in the stage of FIGO butthey have been the routine examination methods in clinical practice [] Many scholars [] have studied the characteristics of those examinations but so far there is no asto which is the best examination method for assessing cervical cancer staging It™s necessary toconduct more studies to identify the role of those tools for early diagnosis of cervical cancerPrevious studies [“] have reported that the transvaginal color Doppler ultrasoundTVCDFI can accurately reflect the size of the lesion the extent of infiltration and the bloodsupply and it is widely used in the detection of cancers in breast and thyroid but not in thecervical parts Besides it is well known that vascular patterns such as density of vessels is different for squamous and adenocarcinomas [] Therefore it™s necessary to show the difference to provide insights into clinical treatment Therefore in order to make clinicians moreclear about the blood supply of cervical cancer and facilitate communication between ultrasound doctors and clinicians we attempted to conduct a preliminary investigation on the correlation between the Adler grade and clinical pathology of cervical cancer to provide insightsinto the diagnosis and treatment of cervical cancer The study design was participantspatients with cervical cancer 2intervention TVCDFI detection and Adler grading 3comparison no applicable 4outcomes Adler secores and antigens CA125 and CA199MethodsEthical considerationsThe study was approved by the Medical Research Ethics Committee of our hospitalNo20180038 and written informed consents were obtained from all the patientsPatientsPatients with cervical cancer diagnosed pathologically in our hospital from January toDecember were selected as study objects in this present study The inclusion criteriawere as follows there were surgical pathology or biopsy detections verifying small cellPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerneuroendocrine tumors by our experienced clinicans and the FIGO cervical cancer stagingwere used for severity differentiation [] no history of other malignant tumors or historyof radiotherapy and chemotherapy complete clinical data no history of cervical surgerysuch as we excluded patients undergone microwave freezing laser electrocautery surgeryThe exclusion criteria were as follows patients with congenital malformations of theuterus patients with coagulopathy patients with severe heart liver and kidney dysfunction patients with inability to cooperate with research or couldn™t followupTVCDFI detectionThe color doppler ultrasound system Philips E80 equipped with ultrasonographic probeDC58D were selected for ultrasound detection Before the detection all patients were askedto empty the rectum and bladder When performing the doppler ultrasound detection thepatients took the lithotomy positions Firstly we performed a transvaginal ultrasound examination of the uterus and bilateral accessory areas to determine the location number size shapeboundary echo of the cervical area and its relationship with surrounding tissues Secondly wechose the color Doppler blood flow imaging mode to observe the blood flow inside and aroundthe cervical area related parameters such the blood flow cancer size have been calculated andcollected The staging and TVCDFI done in the same setting and in different datesAdler grade classificationAdler grade classifications were conducted based on the detected blood flow [ ] We classified the blood flow signal of the lesion into four levels Adler refers to that no obvious bloodflow signal Adler I refers to or small blood vessels with a diameter of mm are detectedAdler II refers to that or small blood vessels are detected Adler III refers to that more than blood vessels or the blood vessels are intertwined into a network are detected The Adlergrade classifications were made by one radiologist and one gynecologist coherently and ifthere were any disparity further discussions were conducted for consensusThe detection of carbohydrate antigens CA125 and CA199Before any drug treatment ml venous blood without anticoagulation in the early morningwere collected from patients the blood specimens were left at room temperature for 15minthen centrifuged it and separated the serum then we stored it at environment with ˚C for further examination The levels of serum CA125 and CA199 were detected by professional stallwith electrochemical luminescence method The electrochemical luminometer and detectionkit used in the test were produced by Roche and the operation process was strictly performedaccording to the instructionsStatistical analysisSPSS statistical software were used for data analysis The measurement data for normal distribution was expressed as mean± standard deviation and the measurement data for skeweddistribution was expressed as median P25 P75 The tumor size CA125 CA199 were compared using the KruskalWallis H rank test of multiple sample comparisons of which Wilcoxon rank test was further used for pairwise comparisons And t tests were conducted in thecancer size between pathological types or squamous cell carcinoma subtypes Spearman correlation analysis was conducted for correlation analysis And receiver operating characteristicROC curves were drawn for evaluating the diagnosis value of Adler grade for cervical cancer[] p005 was considered as being statistically different in this present studyPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerResultsThe ultrasound characteristics of cervical cancerAs Fig presented the ultrasound characteristics differed hugely among the ultrasoundimages with different Alder grades For Alder the cervical morphology was normal andno obvious blood flow signal were found in the images of color Doppler For Alder I thecervical morphology was slightly thickened and more enhanced intracervical echo couldbe collected one or two small spotlike blood flow signals could be detected For Alder IIuneven or thickened cervical echo distribution could be detected and strip blood flow signals at two to four locations could be seen For Alder III parauterine and extrauterineinvasion and blood metastasis could be found and reticular blood flow signals could bedetectedThe distribution of Adler classification and clinical stage of cervical cancerWe identified potential candidates firstly and patients were excluded As Table presented a total of patients were included for data analysis With the increase of Adler severity classification the clinical stage of cervical cancer increased accordinglyFig The Doppler ultrasound images on cervical cancer with different Adler grade a Adler b Adler I c Adler II d Adler III101371journalpone0236725g001PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable The distribution of Adler classification and clinical stage of cervical cancerAdler gradeFIGO gradeGrade IGrade IIGrade IIIIVAdler Adler IAdler IIAdler IIIIn total101371journalpone0236725t001In totalThe comparison on the cancer size CA125 CA199 with Adler grade andpathological parametersAs Table showed the cancer size differed significantly among the different Adler gradesp but there were no significantly differences in the level of CA125 CA199 amongthe different Adler grades all p005 There were significant differences in the level ofCA125 CA199 between the squamous cell carcinoma and adenocarcinoma all p005 nosignificant difference was found in the cancer size between the squamous cell carcinoma andadenocarcinoma p No significant differences were found in the cancer size CA125and CA199 between the keratinized and nokeratinized squamous cell carcinoma all p005Furthermore as Table showed the Adler grade was positively related with the FIGO stagingpathological type and squamous cell carcinoma subtypes of cervical cancer all p005 nocorrelations were found among the Adler grade and the cancer size CA125 CA199 allp005The diagnosis value of Adler grade for cervical cancerAs Fig showed based on FIGO results the area under the ROC curve of the cervical squamous cell carcinoma predicted by Adler grade was p005 and its sensitivityTable Relationship analysis on the cancer size CA125 CA199 with Adler grade and pathological parametersItemsAdler gradeAdler Adler IAdler IIAdler IIItzpPathological typeSquamous cell carcinomaAdenocarcinomatzpsquamous cell carcinoma subtypesKeratinizedNonkeratinizedtzpCasesCancer sizemmCA125[MP25 P75 UmL]CA199[MP25 P75 UmL]±±± ± ± ± ± 101371journalpone0236725t002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable Correlation analysis between Adler grade and clinicopathological parametersClinicopathological parametersFIGO gradeCancer sizeCA125CA199Pathological typesquamous cell carcinoma subtypes101371journalpone0236725t003rpspecificity positive predictive value negative predictive value and Youden index were respectively And based on pathological results the area underthe ROC curve of the cervical squamous cell carcinoma subtype predicted by Adler grade was p005 and its sensitivity specificity positive predictive value negative predictivevalue and Youden index were respectively Thoseresults indicated that Adler grade could provide valuable reference for the diagnosis of cervicalcancerDiscussionsTVCDFI has the advantages of being noninvasive cheap with convenient operation [ ]It can not only clearly display the structure of each layer of the uterus accurately locate andqualitatively diagnose the lesion but also can observe the blood flow of detected area [ ]Dynamic ultrasound is currently one of the most widely used and mature imaging methods inthe diagnosis and treatment of obstetrics and gynecology diseases [] In this present studythe changes of cervical morphology and echo in Adler grade to I were small and Dopplerblood flow signals were less displayed but the blood flow signals in Adler grade II and IIIincreased significantly and the flakelike low echoes were seen inside With the increase ofAdler classification cervical masses continue to increase and color Doppler blood flow signalsare also displayed The results of this present study have indicated that Adler grade is expectedto be useful to reflect the richness of blood flow in cervical cancer which can provide important insights into the diagnosis and treatment of cervical cancerFig The ROC curve on the Adler grade for cervical cancer101371journalpone0236725g002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerThe occurrence proliferation invasion and metastasis of malignant tumors may largelydepend on tumor neovascularization [ ] Cervical cancer has characteristics of rapid proliferation and active cell division and growth which is closely related to the proliferation oftumor vessels in it [] On color Doppler ultrasound images there are often abundant different types of blood flow signals in tumor tissues which correspond to its rich vascular networkand are related to the special structure and blood flow characteristics of tumor blood vessels[ ] The Alder grades are mainly concentrated on the blood flow signals which to someextent can reflect the proliferation of vessels and the characteristics of microvessel densityaround the cervical cancer Previous studies [“] have reported that the blood signals maypredict the ability of tumor invasion and metastasis to a certain extent which is consistentwith the results of our studySquamous carcinoma is more common in the pathological and histological types of cervicalcancer [] We have found that there was correlation between Adler grade and the histopathological type The World Health anization WHO classifies cervical squamous cell carcinoma into two subtypes keratinized and nonkeratinized according to the presence ofkeratinized morphological features in the tissue [] Currently the studies on the relationshipof keratinized cervical squamous cell carcinoma and patient prognosis has been controversialIt™s been reported [“] that compared with nonkeratinized cervical squamous cell carcinoma the survival rate of keratinized cervical squamous cell carcinoma is significantlyreduced and it is not sensitive to radiotherapy and the prognosis is very poor for untreatedpatients or advanced patients Therefore differentiate the various subtypes of squamous cellcarcinoma of cervix is very helpful in clinical managementIn this study Spearman rank correlation analysis showed that Adler grade was positivelycorrelated with squamous cell carcinoma subtypes Therefore this technique is expected to bea reliable indicator for predicting the prognosis of patients with cervical cancerWith the continuous progress of cancer marker research the role of cancer related markerin the diagnosis and treatment of cancers has become increasingly important [ ] Severalstudies have shown that cervical cancer is also closely related to the marker CA125 and CA199It has been reported [“] that the carbohydrate antigens CA125 and CA199 are expressedat high levels in a variety of malignancies In this study the levels of CA125 and CA199 inpatients with adenocarcinoma were significantly higher than those in patients with squamouscell carcinoma The results were consistent with the findings of previous studies [ ] Eventhrough the Spearman correlation analysis shows that there is no correlation between Adlergrade and CA125 and CA199 it may be explained that the samples in this present study is notlarge enough to powerfully detect the difference future studies with larger population samplesare warranted to identify the role of CA125 and CA199 in cervical cancerSeveral limitations must be concerned in this present study Firstly the sample size of thisstudy is not large enough and the clinical staging is relatively rough biases may be existed inthis present study Secondly we did not use the cervical cancer specific markers such as squamous cell carcinoma tumor marker for reference it should be further elucidated in the futurestudies Thirdly it must bring to our attentions that the subjective factors in the Adler classification different operators and different parameter settings may bias the test results Howeverwe have performed related trainings on the TVCDFI before this study to avoid unnecessaryerrors in the process of ultrasound detection and analysisIn the results of this study have favored the value of Adler grade in the diagnosisand treatment of cervical cancer With the continuous development of ultrasound technologyand popularization of clinical ultrasound applications Adler grade should be promoted in theapplication of color Doppler ultrasound for the diagnosis and treatment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerSupporting informationS1 ChecklistDOCXS2 ChecklistDOCXAuthor ContributionsConceptualization Dehong Che Zhirong Yang Jiayin GaoData curation Dehong Che Xuedong Wang Jiayin GaoFormal analysis Dehong Che Zhirong Yang Hong Wei Jiayin GaoInvestigation Dehong Che Zhirong Yang Hong Wei Xuedong Wang Jiayin GaoMethodology Xuedong WangResources Xuedong Wang Jiayin GaoSoftware Dehong CheValidation Dehong Che Jiayin GaoWriting “ original draft Dehong CheReferences Shrestha AD Neupane D Vedsted P Kallestrup P Cervical Cancer Prevalence Incidence and Mortality in Low and Middle Income Countries A Systematic Review Asian Pac J Cancer Prev “ Epub 1022034APJCP2018192319 PMID PubMed Central PMCID PMC5980914 Kalliala I Athanasiou A Veroniki AA Salanti G Efthimiou O Raftis N Incidence and mortalityfrom cervical cancer and other malignancies after treatment of cervical intraepithelial neoplasia a systematic review and metaanalysis of the literature Ann Oncol “ Epub 101016jannonc201911004 PMID Murfin J Irvine F MeechanRogers R Swift A Education income and occupation and their influenceon the uptake of cervical cancer prevention strategies A systematic review J 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Locally Advanced Cervical CancerAsian Pac J Cancer Prev “ Epub 107314apjcp PMID Xie X Song K Cui B Jiang J Yang X Kong B A comparison of the prognosis between adenocarcinoma and squamous cell carcinoma in stage IBIIA cervical cancer Int J Clin Oncol “ Epub 101007s1014701712258 PMID Grjibovski AM Dubovichenko D Saduakassova S Zhatkanbayeva G Omarova G Shalgumbayeva G Incidence mortality and determinants of survival from cervical cancer in Northwest Russia a registrybased cohort study Int Health “ Epub 101093inthealthihx068 PMID Charakorn C Thadanipon K Chaijindaratana S Rattanasiri S Numthavaj P Thakkinstian A The association between serum squamous cell carcinoma antigen and recurrence and survival of patients withcervical squamous cell carcinoma A systematic review and metaanalysis Gynecol Oncol “ Epub 101016jygyno201803056 PMID Kori M Yalcin Arga K Potential biomarkers and therapeutic targets in cervical cancer Insights from themetaanalysis of transcriptomics data within network 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"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 gmbh™as 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 microsurgery˜casemix 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 interventionˆ’mean costs controleffect interventionˆ’ 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
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" the widely recognized anticancer potential of aspirin has created a broad interest to explore theclinical benefits of aspirin in cancer therapy however the current understanding of the molecular mechanismsinvolved in the anticancer potential of aspirin remains limitedmethods cancer stemness assays which contained aldh side population chemoresistance sphere formationand tumorigenesis were performed to validate aspirin function in vitro and in vivo histone modification assay wasperformed to check the effect of aspirin on histone methylation as well as the activity of hdac and kdm6abinhibitor in vivo assay was performed to evaluate therapeutic effects of various inhibitor combination mannersresults in regards to in vitro studies aspirin diminishes cancer cell stemness properties which include reducing thealdh subpopulation side population chemoresistance and sphere formation in three cancer types in regards toin vivo studies aspirin decreases tumor growth and metastasis and prolongs survival in addition our resultsshowed that aspirin inhibits inflammationrelated stemness gene expression especially icam3 identified by a highthroughput sirna platform in regards to the underlying molecular mechanism of action aspirin reduces histonedemethylase kdm6ab expression that mediates histone methylation and suppresses gene expression via a coxindependent manner in regards to therapeutic strategies aspirin combined hdm inhibitors icam3 downstreamsignaling srcpi3k inhibitors or icam3 inhibitor lifitigrast prevents cancer progression in vivos the aforementioned findings suggest a molecular model that explains how aspirin diminishes cancercell stemness properties these findings may provide novel targets for therapeutic strategies involving aspirin in theprevention of cancer progressionkeywords aspirin histone methylation cancer stemness icam3 cox therapeutic strategies correspondence shenwenzhi2011126com1department of pathology and institute of precision medicine jining medicaluniversity hehua road jining 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 stem cell research therapy page of cancer stem cell csc was found as the chief culprit toinitiate tumor occurrence to enhance tumor malignancyand to cause tumor recurrence whereby the maintenanceof cancer cell stemness mainly depends on the tumormicroenvironment or also called the œniche [“] currently the specific properties of csc were identified likehigh aldh1 activity aldehyde dehydrogenase sidepopulation chemoresistance and other cd moleculescd44 cd133 or markers sox2 oct4 nanoglgr5 positive in cancer clinical therapy which targetedhighly tumorigenic cscs may provide new targets orinsight for cancer therapy however unfortunately cscshad demonstrated a relative resistance to conventionalchemotherapy and radiotherapy moreover the cancer cellstemness and the resultant tumor initiationmalignancycould be maintained by the tumorassociated inflammation factors within the tumor microenvironment niche[ ] our previous work presented a mediumthroughput sirna screen platform to identify inflammation genes that regulate cancer cell stemness and obtainedseveral novel candidates agents that target these genesmay inhibit both inflammation and cancer cell stemnessat the same timeaspirin a nonsteroidal antiinflammatory drugiscommonly used as an antipyretic analgesic antiinflammatory and antithrombotic agent [ ] recentobservational and epidemiological studies have shownthat regular prolonged use of aspirin reduces the riskfor several cancers eg colorectal esophageal breastlung prostate liver and skin cancers [“] althoughthe benefits of aspirin for cancer patients have beenwidely appreciated the mechanism remains unclear previous studies attribute the anticancer potential of aspirin to the inhibition of cyclooxygenase2 cox2which is upregulated in various cancer cells [ ] ofnote an increasing body of evidence suggests that aspirin exhibits anticancer effects in a coxindependentmanner histone modification is a reversible process mediated bythe epigenetic enzymes [ ] histone methylation andacetylation are two important chemical modifications thatact in transcriptional activation or inactivation chromosome packaging and dna damagerepair [ ] histone demethylases hdms and histone deacetylaseshdacs are the key enzymes that remove methyl andacetyl groups respectively to regulate gene transcriptionin this regard aspirin was reported to affect hdacs expression and suppress progression of some cancers like aspirin mediates h3k27 acetylation to prevent coloncarcinogenesis and aspirin cooperates with p300 to activate h3k9 acetylation further to promote colorectal cancer cell apoptosis [ ] however the specific rolesand mechanisms of aspirinmediated histone methylationin cancer stemness remains insufficient thus we studiedthe role of aspirin on histone methylation and the attendantand cancerprogressioneffects on cancercellstemnessthe aldh subpopulationour results indicated that aspirin diminishes various cancer cell stemness properties which include reducingside populationchemoresistance and sphere formation in three cancertypes in vitro aspirin inhibits tumor growth and metastasis as well as prolongs survival in vivo aspirininhibits inflammationrelated stemness genes especiallyicam3aspirin reduces histone demethylasekdm6abexpression which mediates histone methylation respectively with a coxindependent manner and aspirin hdm inhibitors aspirin icam3downstream signaling srcpi3k inhibitors and aspirin icam3 inhibitor lifitigrast all reduce cancer progressionin vivo the abovementioned findings demonstrate apromising mechanism of action and potential therapeutic strategy of aspirin in the prevention of cancerprogressionmethodscell culturemdamb231 breast cancer cell and a549 lung cancercell were purchased from atcc hepg2 was obtainedfrom the chinese academy of sciences mdamb231breast cancer cell and hepg2 liver cancer cell were cultured in dmem medium a549 lung cancer cell was cultured in medium allculture media weresupplemented with fbs gibco and were grown at °c in co2 incubators all cells were passaged forless than months before renewal from frozen earlypassage stocks and tested to ensure thatthey weremycoplasma negativecytotoxicity assayaspirin was purchased in sigma cat a2093 and dissolved in dmso mdamb231 a549 and hepg2 cellswere cultured in 96well plate and treated with variousconcentrations and mm for a549 cells and mm for mdamb231 cells and and mm forhepg2 cells of aspirin for h cell activity was testedby applying cck8 kit dojindo china following themanufacturer™s instructionsaldefluor assaythe aldeflour assay kit stemcell technologies vancouver canada was used to measure aldh enzymaticactivity in three cancer cell lines mdamb231 a549and hepg2 in brief cells were treated with aspirin for h and — cells were suspended in aldeflour 0czhang stem cell research therapy page of assay buffer containing aldh1 substrate and incubated for min at °c cells treated with the specific aldh inhibitor deab served as the negative control stained cellswere analyzed on bd facs calibur flow cytometer bdbiosciences san jose ca data analysis was performedusing flowjo software tree star inc ashland orside population assaymdamb231 a549 and hepg2 cells treated with aspirin for h were harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml hoechst dye wasadded at a final concentration of μgml μgmla549 and μgml hepg2 in the presence or absence of μm fumitremin c ftc the followingsteps were described previously [ ]cell apoptosis assaythe mdamb231 a549 and hepg2 cells were treatedwith aspirin and cisplatin ddp μgml for h simultaneously then harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml apoptotic cells were stainedwith propidiumiodide and annexinvfitc bd biosciences flow cytometry analysis was performed by facscalibur cytometer bd biosciences in which a minimum of events were recordedsphere formation assaycells were collected and rinsed to remove serum thendissociated to singlecellsuspension in serumfreedmemf12 medium supplemented with iumlpenicillin μgml streptomycin ngml human recombinant epidermal growth factor hregf ngmlhuman recombinant basic fibroblast growth factorbfgf and b27 supplement invitrogen cells weresubsequently cultured in ultralow attachment 24wellplates at a density of cells per well then treatedwith aspirin all the time until the spheres were formedanimal studyfemale balbc mice at “ weeks were separated randomly into several groups n ‰¥ — 4t1luci cellswere inoculated sc into each mouse at the right axillafor lung metastasis assay at days after injection micewere treated intraperitoneally with aspirin mgkg aspirin mgkg every days and dmso used as thecontrol for chemoresistance assay at days after injection mice were first treated intraperitoneally with cisplatin mgkg then treated with aspirin mgkgaspirin mgkg every days and dmso used as thecontrolnodscid mice at “ weeks were separated randomly into several groups n ‰¥ — a549luci cellswere inoculated sc into each mouse for inhibitor treatment assay days after tumor cells injection micewere first treated intraperitoneally with aspirin mgkgor kdm6ab inhibitor gsk j1 mgkg or src inhibitor a new specific highefficient src inhibitor [ ] mgkg or pi3k inhibitor ly294002 mgkg orlifitegrast mgkg dmso used as the controltumor volume mm3 was measured with calipers andcalculated by using the standard formulalength —width22 the individual measuring the mice was unaware of the identity of the group measured primarytumor tissues were harvested and separated into threeparts one was formalin fixed paraffin embedded andsectioned for ihc staining the other two were brokenby the tissue homogenizer then used for rna trizoland protein ripa lysis buffer extraction animal usecomplied with nankai university and jining medicaluniversity animal welfare guidelines western blottingthe western blot steps were described previously [ ]the special primary antibodies used in this assay arelisted in supplementary table s1 all western data wasthe representative image of three biologically independent repeats the results were quantified using image jsoftware national institutes of health baltimore mdand analyzed by graphpad prism5 software graphpadsoftware san diego ca usanuclear fractionation analysiscells were harvested and the cytoplasmic and nuclearfractions were separated and extracted with an nepernuclear and cytoplasmic extraction kit thermo fisherscientific inc ma usa h3k3me marker proteinswere detected by western blotimmunofluorescencecells grow on glass slides and tumor tissue slices werefixed in paraformaldehyde and labeled with primaryantibodies overnight at °c followed by incubation withspeciesappropriateroomtemperature for h nuclei were stained with dapi andimages were obtained using a leica dm4000 uprightmicroscope or confocal fluorescence microscopy nikontokyo japan [ ]antibodiessecondaryatchromatin immunoprecipitation assaythe assay was performed with an ezzyme chromatinprep kit millipore according to the manufacturer™sprotocol antihistone modification marker antibodieswere used to precipitate dna crosslinked with histone modification markers respectively and normal rabbitigg was used in parallel as a control enriched dna wasthen used as a template to assess the binding intensity of 0czhang stem cell research therapy page of histone modification markers to putative binding sitesin the icam3 promoter primers used in this assay arelisted in supplementary table s2immunohistochemistryimmunohistochemistry was performed on tumor tissuesections from the mice primary antibodies raise againstthe target proteins at a dilution overnight the expression levels of the proteins were evaluated accordingto the percentage of positive cells in each tumor tissuesections the images were recorded by olympus bx51epifluorescent microscopy under a — or — objective olympus co tokyo japan changes in apoptosis in the three cancer cell lines usingthe cell apoptosis assay after cisplatin ddp treatmentour results indicated that apoptosis increases in theaspirintreated groups versus controls and thereby reduces cisplatin chemo resistance fig 1e f in orderto determine the effects of aspirin on cancer cell stemness we next investigated the changes in cell sphere formation in the three cancer cell lines using the sphereformation assay our results showed that sphere formation decreases in the aspirintreated groups versus controls fig 1g h the abovementioned findings suggestthat aspirin diminishes cancer cell stemness propertiesin vitrostatistical analysisall data were analyzed using graphpad prism5 softwaregraphpad software san diego ca usa values wereexpressed as means ± sem p values were calculatedusing a twotailed student™s t test two groups or oneway anova more than groups unless otherwisenoted a value of p was used as the criterion forstatistical significance an asterisk indicates a significantdifference with p two asterisks indicate a significant difference with p and three asterisks indicatea significant difference with p [ ]resultsaspirin diminishes cancer cell stemness properties in vitroin order to establish the proper working concentrationsof aspirin in various cancer cells we determined theic50 of aspirin in a549 lung cancer cells mdamb231breast cancer cells and hepg2 liver cancer cells using acytotoxicity assay our results showed a 107mm ic50in a549 lung cancer cells a 43mm ic50 in mdamb breast cancer cells and a 97mm ic50 in hepg2liver cancer cells fig s1 based on the ic50 we choseworking concentrations of and mm aspirin fora549 lung cancer cells and mm aspirin formdamb231 breast cancer cells and and mmaspirin for hepg2 liver cancer cells in our studiesthe aldh subpopulation decreasesin order to determine the in vitro effects of aspirin oncancer cell stemness we investigated aldh subpopulation changes in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing the aldh staining assay our results indicatedthatin theaspirintreated groups versus controls fig 1a b inorder to determine the effects of aspirin on cancer cellstemness we next investigated the changes in the sidepopulation in the three cancer cell lines using the sidepopulation assay our results indicated that the sidepopulation decreases in the aspirintreated groups versuscontrols fig 1c d in order to determine the effects ofaspirin on cancer cell stemness we next investigated theaspirin diminishes cancer cell metastasis and stemnessproperties in vivoin order to determine the effects of aspirin on cancercell metastasis and stemness in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice seven days after implantation we ip injected themice with mgkg aspirin mgkg aspirin ordmso control group times per week fig 2a ourresults showed thattumor volume decreases in theaspirintreated groups versus the control fig 2b however we found that the body weight did not change inthe aspirintreated groups versus the control fig 2c inaddition we found that the survival time increases in theaspirintreated groups versus control fig 2d with respect to the effect of aspirin on cancer cell metastasiswe found thatlung metastasis decreases in aspirintreated groups versus the control fig 2e“g with respect to the effect of aspirin on cancer cell stemnessproperties we found thatthe immunocytochemicalstaining of sox2 and oct4 stemness markers decreasesin the aspirintreated groups versus dmso controlsfig 2h i the abovementioned findings suggest thataspirin diminishes cancer cell metastasis and stemnessproperties in vivoaspirin reduces cancer cell chemoresistance in vivoin order to determine the effects of aspirin on cancercell chemoresistance in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice eight days after implantation we ip injected themice with mgkg cisplatin mgkg aspirin mgkgcisplatin mgkg aspirin or dmso control groupevery days fig 2j our results showed that tumor volume decreases in the cisplatinaspirintreated versus thedmso control fig 2k however we found that thebody weight did not change in the cisplatinaspirintreated versus the dmso control fig 2l in additionwe found that the survival time increases in the cisplatinaspirintreated groups versus the dmso controlfig 2m we also found that the rate of tumor growth 0czhang stem cell research therapy page of absllec evitisop hdladeabctrl2mm5mm 4mm10mm ctrleddp10ugml2mm5mm 4mm10mm agpehagpeha549ctrl 5mm 10mm ctrl 2mm 4mm sllec evitisop hdlasllec evitisop hdlahepg2fctrl 5mm 10mm slliec ssotpopaa549 slliec ssotpopactrl 5mm 10mm ctrl 2mm 4mm hepg2 slliec ssotpopactrl 5mm 10mm cblockctrl2mm5mm 4mm10mm agpeha549d noitlaupopeds ia549 noitlaupopeds ictrl 5mm 10mm ctrl 2mm 4mm fig see legend on next pagehepg2h noitlaupopeds ictrl 5mm 10mm rebmun erehpsgctrl2mm5mm 4mm10mm agpehhepg2ctrl 5mm 10mm ctrl 2mm 4mm rebmun erehpsrebmun erehpsctrl 5mm 10mm 0czhang stem cell research therapy page of see figure on previous pagefig aspirin restrains cancer cell stemness properties in vitro a aldh staining assay was performed to check aldh subpopulationpercentage in the three cancer cell lines with or without aspirin treatment b statistic results of aldh subpopulation percentage were shown cside population assay was performed to detect sp percentage in three cancer cell lines with or without aspirin treatment d statistic results of sppercentage were shown e facs was performed to detect cell resistance to cisplatin and the percentage of apoptotic cells was shown f statisticresults of apoptosis cells percentage were shown g sphere formation assay was performed to check the cell sphere formation ability in the threecancer cell lines with or without aspirin treatment scale bars μm h statistic results of sphere amounts were shownwas slower in the cisplatinaspirintreated versus thedmso control fig 2n with respect to the effect of aspirin on cancer cell stemness properties we found thatthe immunocytochemical staining of sox2 and oct4stemness markers decreases in the cisplatinaspirintreated groups versus dmso controls fig 2o p theabovementioned findings suggest that aspirin reducescancer cell chemoresistance in vivoaspirin inhibits the expression of inflammationrelatedstemness genes in vitro and in vivoour previously published report established a mediumthroughput sirna screening platform that identifies inflammation genes that regulate cancer cell stemness specifically we identified several novel candidate genesthat decrease oct4 expression and the aldh subpopulation both of which characterize stemness fig 3ain order to determine whether aspirin decreases theexpression of these novel candidate genes to further diminish cancer cell stemness we investigated the expression of novel candidate genes and stemness markerssox2 and oct4 in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing western blot our results showed that icam3ccl16 pde3a prtn3 sox2 and oct4 protein expression decreases in the aspirintreated groups versuscontrols fig 3b fig s2a we also found that icam3ccl16 pde3a prtn3traf6 bcar1 il1a il1bnfkb1 ikbkb sox2 and oct4 mrna expression decreasesin the aspirintreated group versus controlfig 3c moreover in icam3 ccl16 pde3a prtn3traf6 bcar1 il1a il1b nfkb1 sox2 and oct4the protein expression decreasesindicated byimmunofluorescencestainingaspirintreatedmbamd231 fig 3d and a549 cells data not shownversus the controlasin thein order to confirm the above in vitro results we theninvestigated mrna and protein expression in tumorsfrom and 46day aspirintreated miceversus control using qpcr and western blot our resultsdemonstrated that icam3 pde3a prtn3 traf6bcar1 il1a il1b nfkb1 ikbkb sox2 and oct4mrna expression decreasesin the aspirintreatedgroups versus control fig 3e in addition we foundthat icam3 pde3a prtn3 traf6 bcar1 il1ail1b nfkb1 sox2 and oct4 protein expressionsimilarly decreases in the aspirintreated groups versuscontrol fig 3f fig s2b the abovementioned findingsexpression ofsuggesttheinflammationrelated stemness genesin vitro andin vivoaspirin decreasesthataspirin mediates histone methylation to regulate targetgenes expressionin order to determine the mechanism underlying the action of aspirin we explored the regulatory effect of aspirin on histone methylation markers in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using western blot our resultsindicated thatthe expression of h3 trimethylationmarkersie h3k43me h3k93me h3k273meh3k363me and h3k793me increases in the aspirintreated higher concentration groups versus controlfig 4a s3a we also found that the expression of histone demethylases ie kdm6a and kdm6b decreasesin the aspirintreated higher concentration groups versuscontrol fig 4a in addition we studied the protein expression of h3k43me h3k93me h3k273meh3k363me h3k793me and h3 in a549 lung cancercells mdamb231 breast cancer cells and hepg2 livercancer cells using immunofluorescence ourresultsshowed that the protein expression of the h3 methylation markers within the nucleus increases in the aspirintreated groups versus control fig 4b to further support this we extracted the nuclear proteins of eachgroup and detected these h33me markers the resultsalso showed that the expression of h33me markers wasincreased within the nucleusin the aspirintreatedgroups versus control fig 4c s3bin order to identify the role of h3 methylation in regulating selected inflammationrelated stemness genes wemeasured the amount of icam3 dna fragments in h3modification marker pulldowned dnas in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using the chipqpcr assaywe selected icam3 since our previous studies demonstrated that icam mediates cancer cellinflammationand stemness ourtheamount of icam3 dna fragments in the various h3methylation marker pulldowned dnas decreases in alllines fig 4c the abovementionedthree cancer cellfindingssuggesthistoneresults demonstrated thatreducesthataspirin 0cbmcemuovl romutdliavvrus noitcarfctrlasp 50mgkgasp 100mgkgdayscg thgew ydobictrlasp 50mgkgasp 100mgkg days of treatmentectrlasp50 asp100ctrlasp 50mgkgasp100mgkggsedoni ssatsat emgnulfhctrlasp asp ctrlasp asp ——xostcoi sllec xos sllec tcoctrl asp50asp100ctrl asp50 asp100ctrl asp50 asp100zhang stem cell research therapy page of a4t1luci injectionaspirin injectionsurvivalday timej4t1luci injectionaspirin cisplatin 2mgkg injectionsurvivalday kmcemuovl romutctrlcisplatinasp 25cisplatinasp50cisplatin dayslg tihgew ydobctrlcisplatinasp 25cisplatinasp50cisplatin days of treatmentmliavvrus noitcarfncisplatin treatedctrlasp asp50yadyadctrlcisplatinasp 25cisplatinasp50cisplatintimefig see legend on next pageop sllec xoscisplatin treatedctrlasp asp50xostcoctrl asp25 asp50cisplatin treated sllec tcoctrl asp25 asp50cisplatin treated 0czhang stem cell research therapy page of see figure on previous pagefig aspirin suppresses cancer cell metastasis and stemness in vivo a schema of the metastasis model established by subcutaneousimplantation of 4t1luci cells into the 4th pair of mammary fat pad of balbc mice b tumor growth curve of 4t1luci with or without aspirintreatment c the body weight of balbc mice in the course of aspirin treatment d the survival curve of balbc mice inoculated with 4t1luciwith or without aspirin treatment e the representative luciferase images showing the 4t1luci tumors at the primary site and lung metastasissites with or without aspirin treatment f representative he staining images of 4t1luci tumors metastasis to the lung with or without aspirintreatment scale bars lower panel μm g statistic results of metastasis loci of 4t1luci tumors metastasis to the lung with or without aspirintreatment h immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment representativeimages with — magnification were shown scale bars μm i statistic results of sox2 or oct4 positive cells in 4t1luci primary tumors withor without aspirin treatment j schema of the chemoresistance model established by subcutaneous implantation of 4t1luci cells into the 4thpair of mammary fat pad of balbc mice k tumor growth curve of 4t1luci with or without aspirin treatment in the presence of cisplatin l thebody weight of balbc mice in the course of aspirin treatment in the presence of cisplatin m the survival curve of balbc mice inoculated with4t1luci with or without aspirin treatment in the presence of cisplatin n the representative luciferase images showing 4t1luci tumors at theprimary sites with or without aspirin treatment on day before cisplatin administration and day after cisplatin administrationo immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment in the presence of cisplatinrepresentative images with — magnification were shown scale bars μm p statistic results of sox2 or oct4 positive cells in 4t1luciprimary tumors with or without aspirin treatment in the presence of cisplatindemethylase ie kdm6a and kdm6b expressionwhich mediates histone methylation and thereby inhibits gene expression in vitroaspirin mediates h3 methylation to regulate icam3expression in vivoin order to confirm the above in vitro results we nextexamined h3 methylation marker expression in tumorsfrom aspirintreated mice versus control using immunocytochemistry our results demonstrated that the h3methylation marker immunostaining within the nucleusincreases in the aspirintreated group versus controlfig 4d e we also found that the amount of icam3dna fragments in the various h3 methylation markerpulldowned dnas decreasesin the aspirintreatedgroup versus control indicating that icam3 expressionis blocked fig 4f these findings suggest that aspirinmediates h3 methylation and thereby regulates icam3expression in vivoside populationaspirin mediates h3 methylation to regulate icam3expression via a coxindependent mannerin order to determine the role of cox in aspirinmediated h3 methylation and targeted gene expressionwe knocked down cox1 and cox2 expression in a549cells respectively fig 5a s3c and examined thealdh populationand chemoresistance the results showed that the aldh population fig 5b e and side population fig 5c f were decreased in shcox1 or shcox2 cells treated with aspirincompared to shcox1 or shcox2 cells treated withdmso and also the aldh population and side population were decreased in shcox1 or shcox2 cellstreated with aspirin compared to shctrl treated with aspirin moreover the apoptosis was increased in shcox1or shcox2 cells treated with ddp and aspirin comparedto shcox1 or shcox2 cells treated with ddp anddmso ctrl and also the apoptosis was increased inshcox1 or shcox2 cells treated with ddp and aspirincompared to shctrltreated with ddp and aspirinfig 5d g in addition western blot results displayedthat the h3 trimethylation markers were increased andthe histone demethylases ie kdm6a and kdm6bwere decreased in shcox1 or shcox2 cells treated withaspirin compared to shctrl treated with aspirin fig 5hs3d accordingly as the new target genes icam3 expression was decreased in shcox1 or shcox2 cellstreated with aspirin versus shctrl treated with aspirinfig 5i s3e these findings suggest that aspirin mediates h3 methylation and thereby regulates icam3 expression via a coxindependent manneraspirin combined with hdm kdm6ab or icam3signaling inhibitors diminish cancer progression in vivoour previous work proved that icam3 could mediatesrcpi3k signaling to promote cancer cell stemness inorder to investigate the use of aspirin combined withhdm kdm6ab or icam3 signaling inhibitors as thetherapeutic strategies we implanted a549luciferasecells into the fourth fat pad of male nodscid micetwentythree days after implantation we injected ip themice with mgkg aspirin mgkg aspirin mgkg kdm6ab inhibitor gsk j1 mgkg aspirin mgkg src inhibitor mgkg aspirin mgkg pi3kinhibitor ly294002 mgkg aspirin mgkg lifitigrast icam3 inhibitor or dmso control group every days fig 6a our results showed that tumor size andtumor volume decreases in the aspirintreated groupand the aspirin inhibitortreated groups versus dmsocontrol fig 6b c however we found that the bodyweight did not change significantly in the aspirintreatedgroup and the aspirin inhibitortreated groups versusdmso control fig 6din addition we found that the survival time increasesin the aspirintreated group and the aspirin inhibitortreated groups versus dmso control fig 6e these 0czhang stem cell research therapy page of fig see legend on next page 0czhang stem cell research therapy page of see figure on previous pagefig aspirin inhibits the expression of inflammationrelated stemness genes in vitro and in vivo a schematic representation of the sirna screenleft summary of the results from the rnai screen right b western blot examining the expression of inflammatory candidates and stemnessproteins sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirin treatment c quantitative pcr examining the mrnaexpression of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirintreatment d immunofluorescence staining of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cellswith or without aspirin treatment scale bars μm e quantitative pcr examining the mrna expression of inflammatory candidates andstemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treated with aspirin for different survival days f western blotexamining the expression of inflammatory candidates and stemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treatedwith aspirin for different survival dayssuggestresultsthat aspirin combined with hdmkdm6ab or icam3 signaling inhibitors diminishcancer progression in vivo and may serve as the therapeutic strategiesproposed model of aspirin inhibits cancer cell stemnessand cancer progressionbased on our findings we propose the following modelfig aspirin inhibits histone demethylase hdm expression which then mediates histone methylationh3k43me h3k93me h3k273me h3k363meh3k793me respectively these h3 methylations theninhibit the expression of various inflammationrelatedstemness genes previously identified by highthroughputsirna screening il1a il1b icam3 ccl16 traf6pde3a prtn3 nfkb1 ikbkb bcar1 using theicam3 gene as a representative of the inflammationrelated stemness genes by the aspirinmediated h3modifications restrain icam3 promoter activity andcause icam3 expression is inhibited thus aspirin maydiminish cancer cell stemness properties and cancer progression in vitro and in vivo by inhibiting the expressionof various inflammationrelated stemness genes mostinterestingly the above process was not depending oncox expression as the therapeutic strategies aspirincombined various inhibitors suppressed tumor progression effectivelydiscussionthe widely recognized anticancer potential of aspirin aclassical nonsteroidal and antiinflammatory drug hascreated a broad interest to explore the clinical benefitsof aspirin in cancer therapy [“] previous findingsby many investigators have establishe
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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 people™s 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 people™s hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °c for further experiments this research was approved by the ethics committee of lianyungang second people™s 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 dulbecco™s modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0„ƒ 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 real‘time polymerase chain reaction qrt‘pcrthe 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 × 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 japandual‘luciferase 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 phenol“chloroformisoamyl 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 × 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 people™s 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 student™s 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 mir‘‘5pby 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 a03emir‘‘5p 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 mir‘‘5p 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_0020123mir‘‘5pthbs2 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 people™s hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s 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 × 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
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" recently copy number alteration cna of 9p241 were demonstrated in of diffuse large bcelllymphoma dlbcl with gene expression and mutation profiles that were similar to those of primary mediastinallarge bcell lymphoma pmbcl however their cnabased profile and clinical impact still remain unclearmethods multiplex ligationdependent probe amplification were employed to investigate the prevalence of jak2pdl2 amplification in dlbcl and their cnabased pattern of driver genes the clinical outcome and characteristicswere also analyzedresults using unsupervised hierarchical clustering a small group of dlbcl was clustered togetherwith pmbcl as cluster_2 demonstrating amplification of jak2 and pdl2 this subgroups ofdlbcl demonstrated significant higher expression of pdl1 than those with myd88 l265p mutationp andthey exhibited dismal os and pfs as compared with dlbcl_othersp and respectively which issimilar to dlbcl with myd88 l265p mutations dlbcl with amplification of jak2pdl2 exhibits cna pattern that is similar to pmbcl anddemonstrates unfavorable clinical outcome that resembles those with myd88 l265p mutation it is essential toidentify this subgroup of dlbcl who may acquire more benefits from the jak2 and pdl1 signaling inhibitionkeywords diffuse large bcell lymphoma jak2 pdl2 amplification prognosis diffuse large bcell lymphoma dlbcl is a highly heterogeneous disease recently several distinctive geneticsubtypes were identified including schmitz r studymcd bn2 n1 and ezb subtypes and chapuy b study c0 c5 clusters [ ] godfrey j study also correspondence jmyingcicamsaccn lvningcicamsaccn xuemin xue and wenting huang are cofirst authors jianming ying andning lv are cosenior authors1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing chinafull list of author information is available at the end of the identified an unique biological subset of dlbcl withpdl1 gene alterations having high risk features thus the genetics of dlbcl relating to potential therapeutic targets for immune checkpoint inhibitors shouldbe paid much more attention tojanus kinase jak2 programmed cell death ligand pdl1cd274pdcd1lg1 and programmed celldeath ligand pdl2cd273pdcd1lg2 are adjacent to each other on chromosome 9p241 playing keyroles in host immune surveillance amplification of9p241 were frequently seen in celllines of classical and primaryhodgkin lymphoma chl 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 0cxue bmc cancer page of mediastinal large bcell lymphoma pmbcl but much less in dlbcl cell lines ] correspondingly pd1 ligands pdl1 and pdl2transcripts and proteins were more abundant in chl andpmbcl cell lines than that in dlbcl cell lines recently y wang study demonstrated that ofdlbcl had copy number alteration cna of 9p241with a gene expression and mutation profile similar tothose of pmbcl however their cnabased profileand clinical impact still remain unclearin thisthereforestudy we employed multiplexligationdependent probe amplification mlpa to investigate the prevalence of jak2pdl2 amplification indlbcl and their cnabased pattern of driver genesincluding bcl2 cdkn2a and tp53 and we analyzed their longterm survival outcome after treatmentof rchoplike regimemethodscase selectionwe collected consecutive cases of dlbcl and pmbclin our clinical ffpe archives of excisional biopsy database between jan and oct and cases ofdlbcl and cases of pmbcl were found after confirmation one case of dlbcl was diagnosed as pmbclthus cases of dlbcl and cases of pmbcl wereacquired finally see additional file all patients werediagnosed at national cancer centernational clinicalresearch center for cancercancer hospital chineseacademy of medical sciences and peking union medicalcollege according to the revised 4th edition ofthewho classification of tumours of haematopoietic andlymphoid tissues the data regarding treatment andprognosis were acquired by means of medical recordconsultation and telephone conversationmultiplex ligationdependent probe amplification mlpagenomic dna were extracted from formalinfixedparaffinembedded ffpe blocks using qiaamp dnaffpe tissue kit qiagen valencia ca then dnacopy number quantification and myd88 l265p mutation were detected using mlpa kitmrchollandnetherlands the pcr products were detected on anabi genetic analyzer applied biosystems usaand the final result were analyzed using coffalyser software the relative peak ratio prr of probe largerthan was defined as amplification and less than was defined as deletion see additional file geneswhich had two or more probes covering two differentexomes were put into final analysis including jak2 pdl2 mdm2 rel pus10 bcl2 nfatc1 spib foxp1nfkbiz bcl6 prdm1 tnfaip3 cdkn2a ptening1 and tp53 the details of mlpa probes of drivergenes in dlbcl are shown in the online supportingmaterial see additional file true amplification of onegene was regarded only when all probes of this gene exhibited amplification and vice versa see additional file myd88 l265p mutation was identified when theprobe had a high peak myd88 wildtype didn™t show anypeak see additional file immunohistochemistry ihc staining of pdl122c3ihc staining was performed on dako autostainer link asl48 platform each ffpe block were cut at athickness of 4μm and then deparaffinized antigen retrieval were performed using the envision„¢ flex targetretrieval solution at low ph monoclonal pdl1clone 22c3 dako were used as primary antibodyfollowed by incubation with envision„¢ flex mouselinker and then envision„¢ flex hrp reagent finally the ihc was visualized by envision„¢ flex dabeach ihc slide contained a positive controllungcarcinomaihc score of pdl1 were calculated by multiplyingthe percentage of positive cells with mean intensity no staining weak staining moderate staining strong staining which was reported in previous study the results were evaluated by an experienced hematopathologist xueminstatistical analysisthe differences of clinicopathological characteristicsamong different groups were analyzed using chisquaretest fisher exact test or kruskalwallis rank sum testpdl1 ihc score between different groups was analyzedusing wilcoxon test overall survival os and progressfree survival pfs times were defined from the date ofpathologic diagnosis to the date of the event or the lastfollowup the hazard ratio hr of each parameter wascalculated by univariate cox proportional regressionanalysis firstly in which parameters with p wereevaluated together using multivariate cox proportionalregression analysis the survival curve were made according to kaplan“meier procedure the day oflastfollowup was march 1st all statistical analysiswere two sided and p was defined as significanceunsupervised hierarchical clustering was carried outusing euclidean distance and complete method heatmap was plot using pheatmap packageall above statistical analyses were run in r statistic softwareresultsunsupervised hierarchical clustering of cnas of drivergenes and its survival analysis in dlbcl and pmbclpatientsbased on array cgh lenz g study previouslyidentified specific cnas in pmbcl which were different 0cxue bmc cancer page of from abc and gcb of dlbcl abc dlbcls oftenhave cnas in foxp1 nfkbiz cdkn2a cdkn2binf4a bcl2 nfatc1 and spib while gcb dlbclsfrequently harbor cnas in rel pten mdm2 mihg1and ing1 pmbcl often demonstrate cnas of jak2 andpdl2 using unsupervised hierarchical clustering we explored the cnabased pattern of these genes in dlbcl andpmbcl the result showed that a small group of dlbcl was clustered together with pmbcl as cluster_2 with amplification of jak2 and pdl275068fig 1a this subgroup of dlbcl occurred atthe site of cervical lymph node cases gastrointestinal tract cases nasal cavity case and spleen cases fig 1atable 1additional file the frequency of jak2 and pdl2 amplification in the whole cohort of dlbcl were and while both of them were inpmbcl fig 1a see additional file meanwhile all casesin cluster_3 harbored amplification of nfkbiz which is essential for nfκb activation in abc dlbcl but noamplification of nfkbiz was found in cluster_1as to survival dlbcl in cluster_2 demonstrated significant worse os p and pfs p as compared with dlbcl in cluster_1fig 1b howevercluster_1 and cluster_3 didn™t reveal significant differencein survival fig 1b we also analyzed the os and pfs between dlbcl with and without jak2pdl2 amplification and got statistical significance see additional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table fig heatmap and survival analysis based on unsupervised hierarchical clustering and status of jak2pdl1 amplification and myd88 mutationin tcga dataset a heatmap of cnabased profiles of driver genes in dlbcl and pmbcl by using unsupervised hierarchical clustering b survivalcurves and coxregression analysis of os and pfs among three cnabased clusters after rchoplike treatment c status of amplifications of jak2pdl1cd274 and pdl2pdcd1lg2 and mutation of myd88 in dlbcl tcga pancancer atlas from cbioportal [ ] 0ccervical lymphnodefemale high_intermediatehigh_intermediatedlbclnasal cavitymalegcbbreak_apartdlbcldlbcldlbcldlbcldlbcldlbclcervical lymphnodestomachstomachcolondlbclpmbclpmbclpmbclpmbclcervical lymphnodecervical lymphnodemediastinummediastinummediastinummalelow_intermediate non_gcbnormalfemale low_intermediate non_gcbmalemalelowhighnon_gcbgcbnon_gcbnormalnormalnormalnormalfemale low_intermediate non_gcbnormalmalelowfemale female female lowlowhigh_intermediatenanananananormalnormalnormalnormalnormaljak2_amppdl2_amp“““““““““““““xue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265p“myc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didn™t show any significant differences table jak2pdl2 amplification identify a distinctive cnabasedpattern of dlbcl similar to that of pmbclsince dlbcl with jak2pdl2 amplification had less frequency of myd88 l265p mutation our study separateddlbcl patients into three subgroups dlbcl with jak2pdl2 amplification dlbcl_jak2pdl2_amp dlbclwith myd88 l265p mutation dlbcl_myd88_l265pjak2pdl2 amplification norand dlbcl withoutmyd88_l265p mutation dlbcl_others fig 2a basedon the unsupervised cluster result fig 1a one patientwho had both jak2pdl2 amplification and myd88l265p mutation was clustered into cluster_2 thereforethis patient was put into dlbcl_jak2pdl2_amp subgroup accordingly we also analyzed the data when thiscase was included in dlbcl_myd88_l265p subgroupand got the similar result see additional file unlike dlbcl_myd88_l265p and dlbcl_othersdlbcl_jak2pdl2_amp showed a distinctive pattern similar to that of pmbcl with high frequencyof rel and nfkbiz amplifications but no amplification of bcl2 and nfatc1 and no deletion ofprdm1 was found fig 2awith respectto clinicopathologicdlbcl_jak2pdl2_amp tend to be youngerdlbcl_myd88_l265p p hans modelcharacteristicsthantable whileinternational prognostic index ipi riskpdl1 expression in dlbcl with jak2pdl2 amplificationwas significantly higher than that in dlbcl with myd88l265p mutationtotally cases were performed pdl1 22c3 ihc detection including dlbcl_myd88_l265p cases dlbcl_jak2pdl2_amp cases dlbcl_others cases andpmbcl cases the result showed that pdl1 expressionin dlbcl_jak2pdl2_amp was significantly higher thanthat in dlbcl_myd88_l265p p and dlbcl_others p fig 2b and d while no significant difference was found between dlbcl_jak2pdl2_amp andpmbcl p fig 2bjak2pdl2 amplification identify a subgroup of dlbclwith unfavorable survival outcome similar to that ofmyd88 l265p mutationtrying to explore the survival indication of jak2pdl2 amplification and myd88 l265p mutation cases of dlbcls who received rchoplike regimentwith or without surgical resection were enrolled toperformed cox proportional regression analysis of osand pfs the median followup time was monthsrange “ monthsin the univariatecompared withdlbcl_others dlbcls with myd88 l265p mutationhad significantly worse os and pfs p andanalysisas 0cxue bmc cancer page of fig comparison of cnabased pattern pdl1 expression and survival analysis among pmbcl and three subgroups of dlbcl a comparison ofcnabased patterns of driver genes among pmbcl and three subgroups of dlbcl according to the status of jak2pdl2 amplification andmyd88 l265p mutation b comparison of pdl1 expression ihc score among pmbcl and three subgroups of dlbcl c survival curves and coxregression analysis of os and pfs among three subgroups of dlbcl after rchoplike treatment d representative images of he× and pdl1× ihc in dlbcl_jak2pdl2_amp and dlbcl_ myd88_l265p respectively and the same to dlbcls withjak2pdl2 amplification p and respectively meanwhile ipi risk category were significantly associated with os and pfs fig 2c tables and in the multivariate analysis ipi risk category andthree subgroups of dlbcl were put into analysis ascompared with dlbcl_others dlbcl with myd88l265p mutation still showed poor os and pfs p and respectively and the same todlbcl with jak2pdl2 amplification for pfs andos p and respectively meanwhile ipirisk category was still an independent risk predictorsfor os and pfs fig 2c tables and either jak2pdl2 amplification or myd88 l265pmutation are frequently seen in relapserefractory dlbclwith pfs less than yearsdlbcl with pfs less than years was defined as primaryrelapserefractory cases among these cases who treated byrchoplike regime the frequency of jak2 and pdl2amplification were and meanwhilethe frequency of myd88 l265p mutation were dlbcl with either jak2pdl2 amplification ormyd88 l265p accounted for discussiondlbcl presents with a wide spectrum of genetic aberration recently shi study exhibited pdl2 amplification in pmbcl and of dlbcl chapuy demonstrated of 9p241 amplification indlbcl meanwhile dlbcl with pdl1 gene alterations was identified as a unique biological subgrouphaving high risk features y wang study demonstrated that of dlbcl had cna of 9p241 withgene expression and mutation profiles that were similarto those of pmbcl in our study by using unsupervised hierarchical clustering cases ofdlbcl were clustered together with pmbcl as cluster_2 indicating that they shared recurrent cnas theywere enriched for jak2 amplification and pdl2 amplification fig 1a 0cxue bmc cancer page of table comparison of characteristics among pmbl and three subgroups of dlbcldlbclothersmyd88_l265pjak2pdl2_amppmbclpatientsage median range “ “ “ “bmnegativepositiveihc hans™ algorithmgcbnongcbipilowrisklow_intermediatehigh_intermediatehighmyc breakapartnegativepositive p_valueχ2test kruskalwallis rank sum testusing hans model most of dlbcl in cluster_2 werenongcb and tend to be younger than othergroups of dlbcl table which was consistent withprior study therefore coupled with y wang study we confirmed that dlbcl with jak2pdl2 amplification is a unique subgroup resembling the pmbclwith respect to cna patternwith regard to survival increasingly data exhibited thatthe suppression of immune surveillance in dlbcl was associated with poor survival godfrey j study hasdemonstrated that dlbcl with pdl1 gene alterationsshowed high risk features metaanalysis also showedthat pdl1 expression was associated with poor os andadverse clinicopathologic features in dlbcl in y wang study of dlbcl harbored cnaof 9p241 of which were gains and were amplifications and as compared with those who have nogain of 9p241 dlbcl with 9p24 amplification had atrend of better efs while patients with only gain tend tothey didn™thave worse prognosis unfortunatelyshow any statistical significance in our study of dlbcl were found that had cna of jak2when jak2 cna was separated into gain mlpa valuebetween “ and amplification mlpa value as described cases in dlbcl_jak2pdl2_amp group were found that had jak2 gain whichwas slightly lower than that in wang j study asshown in additional file and both dlbcl withjak2 gain and with amplification demonstrated significant poor prognosis as compared with rest of dlbclas shown in additional file more interesting unlikey wang study cases of pmbcl were included in our study as control all of which demonstrating jak2 gains rather than amplifications as shown inadditional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table which was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1cmyd88 l265p is a poor indicator of survival for dlbcl which may lead to primary refractoryrelapsed diseasethis is a gainoffunction driver mutation occurring in of dlbcl but absent in pmbcl [“] inour study the frequency of myd88 l265p in dlbcl andpmbcl were and which were in linewith prior studies [“] of greatinterest myd88l265p mutation occurred less frequently in cluster_2 which was supported by the data tcga pancancer atlas from cbioportal [ ] thus when we divided dlbcl patients into three subgroups dlbcl_jak2pdl2_amp dlbcl_myd88_l265p and dlbcl_others both dlbcl_jak2pdl2_amp and dlbcl_myd88_l265p demonstrated dismal os and pfs with amedian followup of years as compared with dlbcl_others therefore dlbcl with jak2pdl2 amplification 0cxue bmc cancer page of table os in dlbcl treated by rchoplike regimeage ‰¥ bmnegativepositivesiteextranodalnodalihc hans™ algorithmgcbnongcbmyc fish breakapartnegativepositiveipi risk categorylowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clustercluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_jak2pdl2_ampdlbcl_myd88_l265poshr_u95ci “ “ “ “ “ “ “ “ “ “ “ “ “p_valueoshr_m95cip_value “ “ “ “ “hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasn™t put into multivariate analysiswas identified as a poor survival subgroup that is similar todlbcl with myd88 l265p mutationmeanwhile we also compared the cna patterns ofdriver genes among dlbcl_jak2pdl2_amp dlbcl_myd88_l265p dlbcl_others and pmbcl dlbcl_jak2pdl2_amp showed a distinctive pattern similarto pmbcl with high frequency of rel and nfkbizamplifications but no amplification of bcl2 and nfatc1 and no deletion of prdm1 was found the profile ofdlbcl_myd88_l265p was closed to dlbcl_othersshowing relatively high frequency of cdkn2a deletionnfatc1 amplification and bcl2 amplificationin our study of dlbcl_jak2pdl2_ampharbored both jak2 and pdl2 amplifications simultaneouslyindicating that they may also have the pdl1amplification because pdl1 located in the middle ofjak2 and pdl2 at 9p241 thus we hypothesized thatpdl1 expression would be upregulated in this subgroup as what we expected using pdl1 22c3 ihcdetection pdl1 expression in dlbcl_jak2pdl2_in dlbcl_amp was significantly higher than thatmyd88_l265p p and dlbcl_othersp fig 2b and d but not in pmbcl p fig 2b meanwhile pdl1 expression could be 0cxue bmc cancer page of table pfs in dlbcl treated by rchoplike regimeage ‰¥ bmnegativepositivesiteextranodalnodalihc hans™ algorithmgcbnongcbmyc breakapartnegativepositiveipilowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clusterscluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_ jak2pdl2_ampdlbcl_ myd88_l265ppfshr_u95ci “ “ “ “ “ “ “ “ “ “ “ “ “p_value pfshr_m95cip_value “ “ “ “ “hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasn™t put into multivariate analysisenhanced not only by pdl1 amplification but also byjak2 activation [ ] therefore dlbcl with jak2pdl2 amplification was confirmed as an unique subtype that is different from dlbcl with myd88 l265pand othersobjective response rates orr of pd1 blockade therapy was “ in unselected patients with relapsedrefractory dlbcl [ ] the wide spectrum of orrmay be due to high heterogeneity of this subgroupansell sm study demonstrated patients with9p241 alteration in relapsedrefractory dlbcl inour cohort the frequency of jak2 and pdl2 amplification in relapsedrefractory dlbcl were and which were within the range of orr in the prior studies[ ] while patients were found thathad myd88 l265p mutation who may not be suitablefor antipd1 therapy thus the genetic analysis in refractoryrelapsed dlbcl is required for future therapyselection to increase the orr of immune checkpointinhibitorsjak2 amplification could augment the expression of itself and pd1 ligands pdl1 and pdl2 enhancing the 0cxue bmc cancer page of sensitivity to jak2 kinase inhibitor chemical jak2inhibition could reduce the rna transcription and protein expression of pdl1 thus selective inhibitionof jak2 would be a valuable complementary therapy forpdl1 blockadeauthors™ contributionsxx contributed to pdl1 ihc staining clinical followup data analysis andmanuscript writing wh contributed to ffpe tissues collection mlpa detection and clinical followup tq and lg provided experiment guidance anddata interpretation jy and nl contributed to study design coordination discussion and manuscript editing all authors read and approved the finalmanuscriptsjak2pdl2 exhibitsdlbcl with amplification ofpmbcllike cnas pattern and demonstrates unfavorable outcome resembling those with myd88l265p mutation thusit is essential to identify thissubgroup of dlbcl who may acquire more benefitsfrom the jak2 and pdl1 signaling inhibition andjak2 amplification detection by mlpa would be feasible in routine practice meanwhile the difference ofsurvival outcome between our study and wang j study indicated that pmbcllike dlbcl suggested by 9p241 cna could be an intermixed subgroup which required further explorationsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072933additional file mlpa results and clinical followup data the clinicopathological characteristics clinical followup data and mlpa results areshowed in this fileadditional file figure s1 representative results of mlparepresentative results of mlpa are showed in this figureadditional file table s1 the details of mlpa probes of genes indlbcl the locations and lengths of mlpa probes of genes are showedin this tableadditional file the detailed information of dlbcl with jak2pdl2amplification the detailed data about clinicopathological characteristicsmorphology immunohistochemistry and treatments of dlbcl with jak2pdl2 amplification are showed in this fileadditional file figure s2 the os and pfs of dlbcl with or withoutjak2pdl2_amp the os and pfs of dlbcl with or without jak2pdl2_ampadditional file figure s3 comparison of cnabased pattern andtheir survival outcome among pmbcl and three subgroups of dlbclone case of dlbcl with jak2pdl2 amplification and myd88 l265p mutation were included in dlbcl_myd88_l265p group a comparison ofcnabased patterns of driver genes among pmbcl and three subgroupsof dlbcl according to the status of jak2pdl2 amplification and myd88l265p mutation b survival curves and coxregression analysis of os andpfs among three subgroups of dlbcl after rchoplike treatmentadditional file figure s4 the frequencies of jak2 gain andamplification and their survival analysis a the frequencies of jak2 gainand amplification in dlbcl_jak2pdl2_amp and pmbcl b the os andpfs of dlbcl with jak2 gain or with jak2 amplificationabbreviationsdlbcl diffuse large bcell lymphoma pmbcl primary mediastinal large bcell lymphoma mlpa multiplex ligationdependent probe amplificationtcga the cancer genome atlas ipi international prognostic indexffpe formalinfixed paraffinembedded os overall survival pfs progressfree survival hr hazard ratioacknowledgementsnot applicablefundingthis study was partly supported by the beijing municipal science technology commission grant number z151100004015121 the cancerfoundation of china grant number lc2014l13 and cams innovation fundfor medical sciences grant number 2016i2m1001 to perform ffpe tissuescollection and mlpa detection and was partly supported by the cancerfoundation of china grant number lc2018b10 and pumc youth fundand the fundamental research funds for the central universities grantnumber to conduct pdl1 ihc staining and clinical followupand collect fish data of cmycavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its supplementary information filesethics approval and consent to participatethis is a retrospective study that was launched in november the casesenrolled in this project were diagnosed between jan and oct whose ffpe samples were used the data regarding treatment andprognosis were acquired by means of medical record consultation andtelephone conversation thus the need for consent was waived by theindependent ethics committee of cancer hospital chinese academy ofmedical sciences national gcp center for anticancer drugs ncc2015st05consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing china 2department ofpathology national cancer centernational clinical research center forcancercancer hospital shenzhen hospital chinese academy of medicalsciences and peking union medical college shenzhen chinareceived march accepted august referencesschmitz r wright gw huang dw johnson ca phelan jd wang jqroulland s kasbekar m young rm shaffer al genetics andpathogenesis of diffuse large bcell lymphoma n engl j med “chapuy b stewart c dunford aj kim j kamburov a redd ra lawrencems roemer mgm li aj ziepert m molecular subtypes of diffuse largeb cell lymphoma are associated with distinct pathogenic mechanisms andoutcomes nat med “godfrey j tumuluru s bao r leukam m venkataraman g phillip jfitzpatrick c mcelherne j macnabb bw orlowski r pdl1 genealterations identify a subset of diffuse large bcell lymphoma harboring a tcellinflamed phenotype blood “green mr monti s rodig sj juszczynski p currie t o'donnell e chapuy btakeyama k neuberg d golub tr integrative analysis reveals selective9p241 amplification increased pd1 ligand expression and furtherinduction via jak2 in nodular sclerosing hodgkin lymphoma and primarymediastinal large bcell lymphoma blood “ wang y wenzl k manske mk asmann yw sarangi v greipp pt krull jehartert k he r feldman al amplification of 9p241 in diffuse large bcell lymphoma identifies a unique subset of cases that resemble primarymediastinal large bcell lymphoma blood cancer j 0cxue bmc cancer page of lenz g wright gw emre nc kohlhammer h dave ss davis re carty slam lt shaffer al xiao w molecular subtypes of diffuse large bcelllymphoma arise by distinct genetic pathways proc natl acad sci u s a“swerdlow sh campo e harris nl jaffe es pileri sa stein h thiele j whoclassification of tumours of haematopoietic and lymphoid tissues revised4th edn lyon iarc cerami e gao j dogrusoz u gross be sumer so aksoy ba jacobsen abyrne cj heuer ml larsson e the cbio cancer genomics portal anopen platform for exploring multidimensional cancer genomics datacancer discov “gao j aksoy ba dogrusoz u dresdner g gross b sumer so sun yjacobsen a sinha r larsson e integrative analysis of complex cancergenomics and clinical profiles using the cbioportal sci signal pl1 nogai h wenzel ss hailfinger s grau m kaergel e seitz v wollertwulf bpfeifer m wolf a frick m ikappabzeta controls the constitutive nfkappab target gene network and survival of abc dlbcl blood “shi m roemer mg chapuy b liao x sun h pinkus gs shipp ma freemangj rodig sj expression of programmed cell death ligand pdl2 is adistinguishing feature of primary mediastinal thymic large bcelllymphoma and associated with pdcd1lg2 copy gain am j surg pathol“ qiu l zheng h zhao x the prognostic and clinicopathological significanceof pdl1 expression in patients with diffuse large bcell lymphoma a metaanalysis bmc cancer moelans cb monsuur hn de pinth jh radersma rd de weger ra vandiest pj esr1 amplification is rare in breast cancer and is associated withhigh grade and high proliferation a multiplex ligationdependent probeamplification study anal cell pathol amst “fernandezrodriguez c bellosillo b garciagarcia m sanchezgonzalez bgimeno e vela mc serrano s besses c salar a myd88 l265p mutation isan independent prognostic factor for outcome in patients with diffuse largebcell lymphoma leukemia “ ngo vn young rm schmitz r jhavar s xiao w lim kh kohlhammer h xuw yang y zhao h oncogenically active myd88 mutations in humanlymphoma nature “ dubois s viailly pj bohers e bertrand p ruminy p marchand vmaingonnat c mareschal s picquenot jm penther d biological andclinical relevance of associated genomic alterations in myd88 l265p andnonl265pmutated diffuse large bcell lymphoma analysis of casesclin cancer res “ gupta s cheville jc jungbluth aa zhang y zhang l chen yb tickoo skfine sw gopalan a alahmadie ha jak2pdl1pdl2 9p241amplifications in renal cell carcinomas with sarcomatoid transformationimplications for clinical management mod pathol “ ansell sm minnema mc johnson p timmerman jm armand p shipp marodig sj ligon ah roemer mgm reddy n nivolumab for relapsedrefractory diffuse large bcell lymphoma in patients ineligible for or havingfailed autologous transplantation a singlearm phase ii study j clin oncol“lesokhin am ansell sm armand p scott ec halwani a gutierrez mmillenson mm cohen ad schuster sj lebovic d nivolumab inpatients with relapsed or refractory hematologic malignancy preliminaryresults of a phase ib study j clin oncol “ hao y chapuy b monti s sun hh rodig sj shipp ma selective jak2inhibition specifically decreases hodgkin lymphoma and mediastinal largebcell lymphoma growth in vitro and in vivo clin cancer res “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"Cardiac arrhythmias Atrial fibrillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular fibrillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ‚ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are afflicted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [“] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial fibrillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular fibrillation VPCs ventricular premature complexes VT ventricular tachycardia ˆ—Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ “ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial fibrillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with confirmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular fibrillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial fibrillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular fibrillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ‚ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insufficiencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia ‚am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ‚ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular fibril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial fibrillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial fibrillation According to a recent survey of electrophysiology professionals atrial fibrillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using specific AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the first months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insufficiency and the systemic ‚am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients afflicted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with confirmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an ‚am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range “ of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the first documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is significantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the final weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained significantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as definite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and fluoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary findings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpause“dependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeficiency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus fingolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides fluoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to significant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs nificant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ‰¥ ms Changes in ‰¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ‰¥ ms or QTc increase of No patients manifested TdP ‰¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signifi cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ‰¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no significant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ‰¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identified only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ‰¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of significant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this specific population HCQAZM could not be initiated or had to be interrupted in ‰¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the first manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or œforme fruste  of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19“specific risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use ˆšof various formulas among which the Bazett™s correction formula is most commonly used QTc QT RRsec QTc is defined as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTc“QRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F
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pancreatic cancer is a devastating malignancy with a 5year relative survival rate of only “ dependenton the geographical location surveyed [“] with these statistics exhibiting only modest improvementover the last four decades [“] the median survival postdiagnosis ranges from just “ months forlocally advanced disease and “ months for metastatic disease it was estimated by the world healthanisation that pancreatic cancer is currently the 7th leading cause of cancerrelated death being responsible for over deaths worldwide in with incidence increasing pancreatic cancerhas been predicted to be the third leading cause of cancerrelated death in the european union by and the second leading cause of cancerrelated death in the united states of america and germanyby several factors contribute to the poor survival of pancreatic cancer patients a current lack of reliablediagnostic markers that would enable early screening coupled with largely nonspecific symptoms ofdisease results in over of patients presenting with metastatic disease at diagnosis this subgroupof patients have limited therapeutic options and are thus typically administered palliative chemotherapyaimed at prolonging survival and reducing symptoms during endoflife care [“] moreover whilstapproximately “ of patients present with localised disease that is eligible for potentially curativesurgery disease recurs in over of patients postresection ultimately these factorsculminate in more than of patients diagnosed with pancreatic cancer succumbing to disease these harrowing statistics highlight that despite research efforts there remains a lack of understandingof the pathogenesis of disease which in turn limits the development of new therapeuticsreceived march revised july accepted august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211pancreatic ductal adenocarcinomapancreatic ductal adenocarcinoma is the predominant pancreaticmalignancypancreatic cancer can arise from either the endocrine or the exocrine region of the pancreas tumours arising fromthe exocrine compartment are termed pancreatic ductal adenocarcinoma pdac and account for over of allpancreatic cancers pdac develops via a stepwise progression from normal tissue through to invasive lesions which is associated withdistinct morphological characteristics [“] it has been proposed that this process begins with a phenomenontermed acinartoductal metaplasia adm which is a normal homeostatic mechanism whereby acinar cells transdifferentiate into a ductal phenotype in response to particular stimuli however if compounded by an oncogenic˜hit™ cells are pushed towards a pathogenic phenotype that develops into pancreatic intraepithelial neoplasia panin[“] disease progresses through preinvasive stages termed panin1a panin1b panin2 and panin3 priorto invasive pdac this progression is associated with increasing nuclear atypia whereby the nuclei are no longerpositioned basally and loss of normal architecture as cells become more papillary in nature with panin3 lesionsdemonstrating increased mitoses as disease progresses to pdac cells become invasive and breach the basement membrane growing through the extracellular matrix and metastasising to distant ans figure less common precursor lesions include intraductal papillary mucinous neoplasms ipmns and mucinous cysticneoplasms mcns that also develop through multistep processes whilst they share some common featureseach lesion is morphologically and genetically distinct in contrast with panins that form within small ducts ipmnsdevelop within the primary or secondary branches of the main pancreatic duct whilst mcns lack ductal involvement an ‚ammatory tumour microenvironment contributes to pdacpathogenesisan archetypal feature of pdac is the development of extensive stromal networks within the tumour microenvironment tme that can account for up to of the total tumour volume [“] this unique characteristic drives theinflammatory nature of pdac that contributes to its aggressive phenotype desmoplasia is driven by pancreaticstellate cells pscs and cancerassociated fibroblasts cafs that upon activation produce a range of extracellularmatrix ecm components such as collagen laminin and fibronectin which in turn form a physical barrier preventing the penetration of therapeutics [“] though pscs and cafs have been shown to support cancer metastasis and drug resistance they interact with cancer cells in a bidirectional manner with each promoting the survivalgrowth and proliferation of the other [“] quiescent fibroblasts are able to differentiate into two unique subtypes termed myofibroblastic cafs mycafs and inflammatory cafs icafs these two subtypes are distinct whereby mycafs express high levels of αsmooth muscle actin αsma and are located adjacent to cancercells while icafs express low levels of αsma and instead secrete high levels of inflammatory mediators including il6 and are distributed distant from cancer cells within desmoplastic tumour regions broadly mycafsappear to have roles in epithelialtomesenchymal transition emt and ecm remodelling whilst icafs appear tobe involved in inflammation and ecm deposition a third less abundant subtype termed antigenpresentingcafs apcafs has more recently been defined these cells express low levels of both αsma and il6 andinstead express high levels of major histocompatibility complex class ii mhcii and related genes as such allthree subtypes are transcriptionally and functionally distinctthe wider tme contains a plethora of other cell types including endothelial cells tumourassociated macrophagestams and neutrophils tans mast cells regulatory tcells myeloidderived suppressor cells mdscs dendriticcells natural killer nk cells and nerve cells interactions between various cells within the tme can driveeither proor antitumorigenic functions of others for example cancer cells can induce pscs to secrete inflammatorycytokines that drive immune cells towards an immunosuppressive phenotype and also form a positive feedback loopby increasing the tumorigenic potential of cancer cells the ecm itself has also been suggested to modifypsc behaviour in particular that ecm stiffness promotes the mycaf phenotype indicated by increased αsmaexpression this results in substantial complexity that ultimately determines tumour phenotype the components of the microenvironment modify tumour behaviour through the production of cytokines growthfactors and other signalling molecules that predominantly drive a proinflammatory and immunosuppressive program that enhances pdac tumour growth and progression [“] figure the ability of the tme toinhibit therapeutic efficacy through both molecular mechanisms and physical fibrotic barriers contributes to the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211figure our current understanding of the contribution of il6 family cytokines to panin and pdac developmentpreinvasive panin lesions develop from normal ductal epithelia through panin stages 1a 1b and to stage invasive andormetastatic pdac this process is associated with acinartoductal metaplasia adm early in disease combined with an accumulation of oncogenic mutations most common mutations are indicated a number of cells within the tumour microenvironment havebeen shown to secrete il6 family cytokines which in turn results in the activation of a protumorigenic signalling cascade a betterunderstanding of the relationship between each of these cells within the tumour microenvironment may reveal new therapeuticopportunities to manage cancer progressionintrinsic resistance of disease thus dual targeting of cancer cells and the tme may be required to induce afavourable therapeutic response although this poses a signficant scientific and clinical challenge [“]molecular basis of pathogenesispdac development is associated with accumulation of mutationsthe progression of tumorigenesis through panin and pdac stages is associated with the stepwise accumulation ofspecific genetic mutations that drive malignant transformation the most frequent genetic alteration is an activatingkras point mutation codon that occurs early on in tumour development and is detected in over ofpdac tumours [“] mutations in kras have been shown to drive development of precursor panin lesions andwhen combined with an appropriate tumour suppressor mutation these lesions progress to invasive or metastaticpdac figure patient tumours harbouring wildtype wt kras often carry activating mutations indownstream effector molecules such as braf or pik3ca the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211inactivation of a range of tumour suppressor proteins is also common including mutations in tp53 cdkn2aand smad4 in approximately and of tumours respectively whilst each mutation has uniquemechanistic outcomes all three proteins are either directly or indirectly involved in the regulation of the g1s cellcycle checkpoint analysis of patient tumours indicates that two or more of these mutations often occur together withcdkn2a mutation being combined with either tp53 smad4 or both usually in the background of kras mutation this suggests that by disrupting this checkpoint cancer cells are able to overcome inhibitory mechanismsallowing continued progression to invasive diseaseunbiased sequencing efforts have also enabled identification of low prevalence pdac mutations observed in lessthan of cases these include mutations in genes involved in chromatin modification kdm6a dnadamage repair atm and other tumourrelated processes such as growth tgfbr1 or tgfbr2 furthermore it is important to note that technical advances are continuously uncovering epigenetic mechanisms thatfurther modulate the pdac transcriptional landscape and ultimately influence disease heterogeneity and tumourprogression molecular subtypesthe pdac epithelial compartment is typically divided into two subtypes including a classical subtype exhibiting anepitheliallike expression profile and a squamous or mesenchymallike subtype an additional third exocrinesubtype is outlined in some analyses and is characterised by a gene expression profile related to digestive enzymeproduction the classical or epithelial subtype has also been further divided into a pancreatic progenitor andan immunogenic subtype whereby the immunogenic subtype is distinguished by significant immune infiltration andassociated gene programmes though there is no consensus on which classification system will allow the mostvaluable stratification of patients the mesenchymal subtype is invariably associated with a poor prognosis the stromal compartment has also been classified into either normal or activated subtypes reflecting the proandantitumorigenic capabilities of the tme with the activated subtype associated with reduced survival this isparticularly valuable as the extensive heterogeneity of pdac complicates clinically relevant stratification of patientsthus the identification of molecularly unique subtypes may enable development of tailored therapeutic regimensthat will provide improved clinical outcomescurrent treatment optionsregardless of disease stage at time of diagnosis patients have relatively limited treatment options for the majorityof patients disease will be locally advanced or metastatic disqualifying them from undergoing potentially curativesurgery in these cases patients are typically offered chemotherapy with palliative intent [“]surgery provides the only potentially curative treatmentsurgical resection remains the only potentially curative treatment option due to minimal efficacy of standardofcarechemotherapy and radiotherapy due to its aggressive nature the majority of patients present to clinic with locallyadvanced or metastatic disease with only “ of patients presenting with localised tumours that are eligiblefor surgical resection even for those able to undergo surgical intervention over of patients relapsepostresection with median survival improving to months and 5year relative survival rate increasingmodestly to “ the use of neoadjuvant therapy is generally reserved for borderline resectable disease inan effort to enable patients to become eligible for surgery however a range of recent trials have shown improved clinical outcomes including overall survival for neoadjuvant treatment of patients with resectable tumours following surgical resection patients are typically treated with adjuvant gemcitabinebased chemotherapy although a recent study showed improved diseasefree survival and overall survival with a modified folfirinoxtherapy combination of oxaliplatin irinotecan leucovorin and fluorouracil radiotherapy provides variable clinical outcomewhilst the use of radiotherapy and chemoradiotherapy combination chemo and radiotherapy in the neoadjuvantand adjuvant settings have been investigated there remains a lack of consensus regarding therapeutic benefit this is due to issues such as insufficient radiation dose and low participant numbers as well as low uptake of moderntechniques in the neoadjuvant setting preliminary studies reported reduced lymph node positivity and rates oflocal recurrence for chemoradiotherapy compared to surgery with adjuvant chemotherapy however the useof radiotherapy in the palliative setting was reported to modestly reduce overall survival more recent studiesusing ablative doses of radiation have shown a survival benefit highlighting that technological advancements mayprovide an avenue for improved clinical outcomes following radiotherapy these contrasting results highlight the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211need to determine which subset of patients may benefit from the inclusion of radiotherapy approaches in standardtreatment regimenschemotherapy remains the cornerstone of treatmentdespite modest improvements in overall survival palliative chemotherapy remains the standard treatment optionfor patients with locally advanced or metastatic disease gemcitabine monotherapy has been the mainstay treatmentfor pancreatic cancer since when it was demonstrated to improve median survival by just over month compared with fluorouracil within the last decade there have been some further improvements in clinical outcomewith combination chemotherapies gemcitabinenabpaclitaxel and folfirinox providing median overall survivalbenefits of and months respectively compared with gemcitabine alone although folfirinox treatment resulted in a lower percentage of patients experiencing reduced quality of life it also had increased toxicity andadverse events thus preventing its administration to patients with multiple comorbidities therapeutic resistance remains a significant barrier to patient survivaldespite advances in chemotherapeutic options treatment efficacy and patient prognosis remain poor due to the inherent therapeutic resistance of pancreatic cancer it has been proposed that this drug resistance may be driven by thetme including changes to cytokine signalling and metabolic pathways [“] this intrinsic resistance is demonstrated by patients experiencing similar overall survival for chemotherapy treatment “ months compared withbest supportive care “ months which encompasses the use of palliative surgery psychological support painmanagement and other methods of symptom control whilst a range of targeted treatments such as egfr orcheckpoint inhibitors have been trialled with or without chemotherapy they have shown limited success [“]emerging roles for the il6 family of cytokines in pdaccytokines are soluble molecular messengers that enable efficient communication between a range of cell types andhave been recognised to be major contributors to the growth and metastasis of cancers [“] in pancreatic cancer cytokines mediate signalling between cancer cells and the cells of the tme including pscs cafs endothelialcells and a range of immune cells including macrophages mast cells neutrophils and regulatory tcells [“]it is the specific signalling pathways active within this community of cells that dictates the balance of pro andantitumorigenic functions the il6 family of cytokinesthe interleukin il6 family of cytokines includes il6 il11 leukaemia inhibitory factor lif oncostatin mosm ciliary neurotrophic factor cntf cardiotrophin1 ct1 cardiotrophinlike cytokine clc neuropoietin np il27 and il31 [“] these cytokines are grouped as they share structural similarity forming a fourαhelical bundle termed helices ad with an upupdowndown topology il6 and il11 utilise a hexameric signalling complex consisting of two molecules each of the cytokine αreceptoreither il6r or il11r respectively and βreceptor glycoprotein gp130 [“] il6 and il11 are ableto signal via two distinct mechanisms termed classic and transsignalling classic signalling involves the formation of a complex including membranebound il6r or il11r with gp130 and the respective cytokine converselytranssignalling utilises soluble il6r or il11r molecules which are able to form a signalling complex with gp130and the respective cytokine [“] in this way classic signalling relies on the responding cell™s intrinsic expressionof il6r or il11r whilst transsignalling is able to activate any cell expressing gp130 lif osm il27 and il31 signal through trimeric complexes with a single cytokine molecule engagingthe respective receptor lifr osmr il27r wsx1 or il31r and either gp130 or osmr for il31[“] cntf ct1 clc and np form tetrameric signalling complexes composed of one cytokinemolecule one lifr one cntfr and one gp130 receptor in each case the active signalling complex consists of two chains that are signalling competent with a combination of either gp130 lifr osmr il27r or il31r the requirement for multiple receptor subunits means that although gp130 is ubiquitously expressed the expression of other receptor subunits dictates the ability for any given cell to respond to cytokine as signalling initiationrequires the presence of cytokine and a compatible receptor complex figure 2asignalling complex assembly leads to transphosphorylation and activation of receptorassociated janus tyrosinekinases jaks largely jak1 and to a lesser extent jak2 and tyk2 in the case of gp130mediated signalling this results in phosphorylation of the cytoplasmic domain of gp130 at tyrosine y and phosphotyrosine py and of gp130 provide docking sites for signal transducer and the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211figure il6 family cytokine signalling pathwaya schematic representation of the stepwise binding process for the il6 family members with il6 as an example the site interaction involves cytokine binding to the respective receptor with the site interaction generally between the cytokine and thecommon gp130 receptor chain finally site interactions involve formation of the final active signalling complex in this case formation of the il6il6rgp130 hexameric complex b general outline of the il6 family cytokine signalling pathway formation ofan active hexameric complex leads to activation of jaks with subsequent activation of the stat3 mapk and pi3k pathways leftthis results in upregulation of the negative regulator socs3 as well as a range of inflammatory and protumorigenic moleculesthe pathway is inhibited by socs3 pias3 and ptps via dephosphorylation ubiquitinmediated proteasomal degradation andsumoylation right the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211activator of transcription stat molecules leading to their subsequent phosphorylation by jak1 and formation ofactive stat dimers [“] phosphorylated stat pstat dimers then translocate to the nucleus and modulatetarget gene expression including upregulation of a range of genes involved in inflammatory and protumorigenicpathways [“] figure 2b broadly these stat3regulated genes can be categorised into pathways associatedwith inhibition of apoptosis increased cell proliferation modulation of immunity and inflammation increased angiogenesis and increased invasive and metastatic potential [“]although jakstat signalling is the predominant pathway activated downstream of il6 family cytokines themitogenactivated protein kinase mapk and phosphoinositide 3kinase pi3k pathways can also be activated the mapk pathway has been suggested to be activated by a src homology domain 2containing phosphatase shp2mediated mechanism whereby shp2 is recruited to py759 on gp130 allowing jakmediated phosphorylation of shp2 this promotes association with the adaptor protein growth factor receptor bound protein grb2 leading to activation of the gprotein ras via son of sevenless sos with a subsequent phosphorylationcascade including raf mek and erk12 activity following this a mapkdependent phosphorylationevent leads to the recruitment of grb2associated binding protein gab1 to the plasma membrane where gab1 issuggested to act as a scaffold or adaptor protein to allow binding of pi3k and shp2 leading to activation of the pi3kand mapk pathways respectively figure 2bthe suppressor of cytokine signalling socs3 is largely responsible for regulation of signalling and is directlyupregulated by stat3 socs3 contains an sh2 domain allowing it to bind to py residues within the gp130receptor with preferential binding to y759 once bound socs3 recruits an e3 ubiquitin ligasecomplex containing cullin5 rbx2 and adaptors elongin b and c via its socs box domain this complexubiquitinates the gp130 receptor inducing its internalisation and targeting it for proteasomal degradation [“]and is also able to ubiquitinate jak2 in vitro socs3 also mediates direct inhibition of the kinase activityof jak12 via its kinase inhibitory region [“] thus socs3 is able to downregulate il6 family cytokinesignalling pathways through two distinct mechanismsthe phosphotyrosine phosphatases ptps and protein inhibitors of activated stats piass also limit the strengthand duration of cytokine signalling a range of ptps including shp2 are responsible for dephosphorylatingtyrosine phosphorylated substrates including jaks stats and other shp2 molecules pias3 preferentially binds pstat3 and inhibits activity either by preventing stat3 interaction with dna by recruiting transcriptional repressors to stat3 target genes or by sumoylating stat3 to prevent its activity figure 2binterleukin in pdacelevation of serum il6 is a negative prognostic marker in human pdacserum il6 levels were increased in pdac patients compared with healthy patients [“] or those withchronic pancreatitis and were also increased in patients with metastatic pdac compared to thosewith locally advanced disease [“] moreover elevated serum il6 positively correlated with increased diseaseburden weight losscachexia and metastasis [““] however there are conflicting observations inthe literature regarding il6 and cachexia although increased serum il6 levels correlate with increased disease stage and in metastatic patients correlates with poor overall survival as such it has been suggestedthat il6 may be a superior marker for diagnostic and prognostic purposes compared with the standard creactiveprotein crp carcinoembryonic antigen cea and carbohydrate antigen ca199 markers il6 is expressed within the tmell6il6 was overexpressed in human pdac tumours in comparison with adjacent normal tissue whilstthis tumourspecific elevation has been correlated with reduced survival in some studies othersshowed no significant correlation with survival similar to the data available in the cancer genome atlastcga dataset for both il6 and il6r figure 3ab the tcga comprise aggregate sequencing data which doeshave limitations regarding interpretation of contributions of individual cell populations to disease outcome howeverit remains a widely used resource for exploratory investigations however overexpression of il6 has been observedat the mrna and protein level in the pancreata of pdac mice with il6 expression increasing with agewhich is indicative of disease stage in these models despite the presence of il6 in tumours primary human and commercial pancreatic cancer cell lines have been reported to exhibit variable expression levels of il6 and secreted cytokine albeit consistently higher than normal pancreatic ductal epithelial cells in an anoid model minimal il6 was expressed by pancreaticcancer cells pccs or pscs in monoculture however in coculture pccs expressed only il6ra whilst icafs expressedhigh levels of il6 with this activating stat3 within pccs icafs also demonstrate an upregulation of the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211figure il6 family cytokine expression in pdac patientsoverall survival for patients with high top quartile and low bottom quartile level expression of a il6 b il6r c il11 d il11re lif f osm g cntf h ctf1 ct1 i clcf1 clc and j il27 n per group data and graphs obtained fromoncolnc using data from the cancer genome atlas tcga statistical significance determined by mantelcox logranktest the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211the jakstat pathway with expression of il6 being dramatically increased in vitro when incubated with pcc conditioned media indicating that soluble factors trigger il6 production more recently pccderived il1αhas been shown to induce autocrine lif secretion and thereby promote the icaf phenotype including activation ofthe jakstat signalling pathway and il6 production in addition tams have been identified as producers of il6 in pancreatic cancer by correlative immunohistochemistry and expression analysis of isolated cell populations production of il6 by tams was shownto influence tumour development via bonemarrow chimeras as mice reconstituted with il6 knockout ko il6myeloid cells developed lowgrade panins whilst those reconstituted with il6 wt cells developed panin3 lesions il6 is a driver of pdac pathogenesisboth in vitro and in vivo studies suggest that the presence of il6 in the tme can drive activation of stat3 with il6 inhibition reducing stat3 phosphorylation this il6stat3 program has been proposed tobe a driver of pdac pathogenesis by enhancing tumour initiation and progression angiogenesis regulation of cytokine expression and immune cell behaviour resistance to apoptosis and promotion of metastasis [“] in aninducible krasdriven mouse model genetic deletion of il6 resulted in a reduction of adm and panin formationwhen kras mutation was initiated embryonically compared with controls suggesting a role for il6 in tumour initiation this was also observed in a constitutive kras mutant model where genetic deletion of il6 preventedtumour initiation in vivo with a reduction in the number of panin and lesions interestingly oncogenickras and hypoxic conditions both features of pdac tumours were shown to induce il6 production perhaps representing a feedforward pathway enhancing tumorigenesis however il6 is notabsolutely required for panin formation as induction of kras mutation at weeks of age in conjunction with anexperimental pancreatitis model drove formation of panin lesions that were not significantly different between il6wt and ko mice il6 mice exhibited reduced tumour progression with decreased proliferative capacity of both cancer and stromal cells enabling regression of precursor lesions furthermore this inhibition of tumour progression by il6deletion was due at least in part to the reversal of adm with ductal cells reverting to an acinarlike phenotype increased apoptosis of cancer and stromal cells was also shown to contribute to this reduced tumour progression as demonstrated by appropriate immunohistochemical analyses with upregulation of proapoptotic anddownregulation of antiapoptotic bcl2 family members this is mirrored by in vitro data whereby il6 stimulation increased the expression of antiapoptotic bcl2bcl2 and bcl2l1bclxl with blockade of il6signalling or stat3 activation inducing apoptosis collectively these data suggest that whilst il6 contributes it is not required for pdac initiation and progressionthe process of angiogenesis supports tumour growth and progression by enabling adequate blood supply whichis enhanced by il6 signalling upon il6 stimulation pdac cell lines upregulate key angiogenic factors such asvascular endothelial growth factor vegfvegf and neurophilin1 nrp1nrp1 with significant correlation observed between the expression of il6r and vegf on human pdac sections il6inducedupregulation of vegf correlated with a growth advantage in pccs with both features inhibited by treatment witha jak2 inhibitor another facet of the protumorigenic effects of il6 is the regulation of cytokine expression that enables modulationof the immune system in particular it has been shown that il6 is able to upregulate a type cytokine profile invitro that may inhibit antitumour immunity in disease il6 suppressed the differentiation of human cd14cells into dendritic cells dcs in vitro whilst combination treatment with il6 and granulocyte colonystimulatingfactor gcsf inhibited the ability of dcs to respond to alloantigen a process that is required for dc maturationand antigen presentation where these effects were reversed by blockade of il6 andor gcsf il6 has alsobeen implicated in driving increased apoptosis of type i conventional dcs cdc1s leading to cdc1 dysfunctionearly in tumorigenesis and thereby decreased cd8 tcell activation this notion is further supported by invivo studies whereby genetic deletion of il6 in a krasdriven pdac mouse model exhibited a significant decreasein the percentage of intratumoral cancerpromoting macrophages and mdscs utilising primary human pscsmdsc differentiation was shown to be driven by pscderived il6 in a stat3dependent manner the resultantmdscs were able to suppress tcell proliferation suggesting a role for il6 in promoting an immunosuppressivetme correlative analysis indicates that il6 through the generation of metabolic stress indirectly causes a reductionin the percentage of intratumoral natural killer nk cells cd4 and cd8 tcells in precachectic and cachecticmice this effect was coupled to a reduction in the expression of an array of genes involved in immune cell the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211recruitment and tcell effector function indicating that il6 is able to directly and indirectly modulate immuneregulation to enhance tumorigenesisemt migration and invasion are prerequisite abilities that are required for tumour metastasis stimulation of pccswith il6 modulated the expression of a range of proteins that drive emt and migration including upregulationof snai1snai1 snail snai2 slug cdh2 ncadherin vim vimentin fn1 fibronectin col1a1 collagen and twist2 and downregulation of cdh1cdh1 ecadherin with these effects mitigated by il6 orstat3 inhibition il6treated pccs and preinvasive panins exhibited morphological cha
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" 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 crohn™s 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 crohn™s 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 [“]alkaloids”a class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atoms”are 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 parkinson™sdisease alzheimer™s 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 andalzheimer™s disease in shsy5y cells anatabine alsoreduced the expression of betasecretase ”the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimer™s disease”through 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 œonline 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 2b“c 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 œimmune system œextracellularmatrix anization œprotein localization œmetabolism œhemostasis and œsignal transduction fig 3bof these we further explored œimmune system œextracellular matrix anization and œsignal transductionas 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 œimmune system categoryincludes œadaptive immune system œcytokine signaling in immune system and œinnate immune systemin particular within these immune categories œcytokinesignaling egincluding il6 family signaling andœtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome œsignal transduction category œsignaling byreceptor tyrosine kinasesby rhogtpases œsignaling by transforming growth factortgfbeta family members œmapk family signalingcascades œintegrin signaling œsignaling by erythropoietin and œsignaling by gpcr were found to be significantly impacted online resource online resource within the œextracellular matrix anization category almost all subcategories were perturbed includingœdegradation of the extracellular matrix œelastic fiberformation and œextracellular matrix proteoglycansonline resource online resource œsignalingœdegradation 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 pc1“pc2plane 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 expressionœfold 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 œana 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 œwater dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure œanatabinenicotine 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 œheatmap 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 œanatabinenicotine dss effect captured by the pairwise comparisons œanatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors œdss treatment and œanatabinenicotine exposuretogether with the adjusting twofactor interaction term œanatabinenicotinedss 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 reactomeœimmune system category and for the twofactor œana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ˆ’ ] 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 thetlr“il1r“tnfr 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 mgkgexpressionvalues”from theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six œleadingedge genes”genes of the gene set with the highestœimmunedifferentialsystem œextracellular matrix anization and œsignaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 œsignaltransduction and œimmune system categoriesil6œsignal transduction and œimmune system categoriesmmp13 œextracellular matrix anization categoryserpine1 œsignal transduction and œextracellular matrixanization categories thbs1 thrombospondin œsignal transduction and œextracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase œextracellular matrix anization and œimmunesystem 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 œbeta is the slope of the best interceptfreelinear fit between microarray and qpcr values œr2 is the coefficient of determination measuring the œgoodnessoffit and œpval 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 œimmune responses œsignal transduction and œextracellular 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 ofalzheimer™s 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 alkaloids”in particular isoquinoline alkaloids present in traditional medicineherbs”exertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i
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"development of cancer is a problem that has accompanied mankind for years The growing number of cases emerging drug resistance and the need to reduce the serious side effects of pharmacotherapy are forcing scientists to better understand the complex mechanisms responsible for the initiation promotion and progression of the disease This paper discusses the modulation of the particular stages of carcinogenesis by selected physiological factors including acetylcholine ACh peroxisome proliferatoractivated receptors PPAR fatty acidbinding proteins FABPs Bruton™s tyrosine kinase Btk aquaporins AQPs insulinlike growth factor2 IGF2 and exosomes Understanding their role may contribute to the development of more effective and safer therapies based on new binding sitesINTRODUCTIONThe increasing prevalence of various types of cancers is a global phenomenon that has been occurring widely and affecting increasing numbers of people In over million new cases were reported and statistics show that every fifth man and every sixth woman is at risk for developing cancer [] Carcinogenesis is a multistage process that leads to cancer development It includes three main stages initiation promotion and progression and each is characterized by different processes During the promotion stage the genetic material of cells is damaged and if not detected by repair systems before the next division the change is transferred to newly formed cells Under the control of growth factors initiated cells may move on to the second stage of cancer formation“promotion During that stage mutated cells undergo multiple divisions their growth is uninhibited as they become insensitive to apoptotic signals As a result the tumor increases its mass but the cells remain within one an The formation of metastases occurs in the next phase“progression Metalloproteinases secreted by tumor cells destroy the extracellular matrix This allows them to enter the bloodstream and reach a new an at a considerable distance from the primary lesion with the stream of flowing blood The key step for the survival of migrating cells is adhesion to the attacked an and the formation of new blood vessels thanks to which they will receive the components necessary for development Angiogenesis the formation of new blood vessels is the last element of carcinogenesis and is enhanced by tumor cell derived factors metabolic changes and a decrease in available oxygen Each of these processes is controlled by different signal pathways via proteins that naturally occur in the body Figure Dysregulation of their expression and thus activity both to an excessive and insufficient degree ensures the continuity of carcinogenesis and increases the chances cancer cells survive in a host anism Understanding the role of individual substances and components of the body in the subsequent stages of neoplastic transformation provides a more complete picture of cancer pathogenesis This allows to develop new recommendations to improve the quality and safety of pharmacotherapy which has a direct effect on an improvement in the cancer patients™ quality of life It also provides the basis for research aimed at developing new compounds that are effective weapons in the fight against cancer Therefore there is no doubt that any actions that bring scientists closer to solving the problem of insufficient cancer therapy are sensible and much needed The purpose of this paper was to discuss the role of selected physiological factors in the various stages of neoplastic transformationOncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cCrucial mechanisms involved in carcinogenesisThe carcinogenic process is regulated by many molecular and cellular mechanisms The diversity of signaling pathways exploited during cancer initiation promotion and progression is immense Many of them are common in different cancer types but there are also clearly unique molecular and cellular signaling signatures specific for particular cancers [] Pathways such as AktPI3K RasMAPKERK Wntcatenin JAKSTAT occupy crucial roles in the regulation of cell proliferation apoptosis differentiation angiogenesis cell migration and invasion immunological activities and inflammationPI3K phosphatidylinositol 3kinase is a family of enzymes that after activation by growth factors cytokines and hormones are involved in the conversion of PIP2 phosphatidylinositol45bisphosphate into PIP3 phosphatidylinositol 345trisphosphate [] The latter in turn takes part in recruiting Akt into the cell membrane where it is phosphorylated and activates other agents such as mTOR NFkB Wnt or inhibits factors like Bad p27 to enhance carcinogenesis Dysregulation of this pathway occurs in endometrial [] bladder [] or gastric cancers [] The aforementioned PIP2 can be also transformed by phospholipase C into DAG and IP3 which then activate protein kinase C PKC [] One of the PKC substrates is Raf kinase which is an element of the RasMAPKERK pathway which is involved in the pathogenesis of ovary cancer [] or nonsmall cell lung cancer [] Ras proteins activate Raf which then phosphorylates kinases MEK12 Its final substrate is kinase ERK12 which after transportation into the nuclei regulates transcription factors essential for DNA synthesis and cell cycle progression They condition cell growth proliferation and viability [] Because of that targeting those two cascades as an anticancer therapy seems to be beneficial However it is important to notice that the existing crosstalk between both of the pathways can impede treatment and inhibition of one can strengthen the activity of the other Another component that is also connected with the MAPK family Figure Modulators of individual stages of carcinogenesis Inducers red frames inhibitors green frames M1rec muscarinic receptor M3rec muscarinic receptor Nrec nicotinic acetylcholine receptor COX2 cyclooxygenase2 PPARÎ peroxisome proliferatoractivated receptors gamma Btk Bruton™s tyrosine kinase FABP fatty acidbinding protein LFABP Ltype fatty acidbinding protein AQPs aquaporins IGF2 insulinlike growth factor IGFBP3 insulinlike growth factor binding protein IGFBP5 Insulin Like Growth Factor Binding Protein IGF2BP2 Insulin Like Growth Factor2 mRNA Binding Protein2 IGF2BP3 Insulin Like Growth Factor2 mRNA Binding Protein Oncotargetwwwoncotargetcom 0cis a signaling pathway conducted by p38MAPKs This subfamily contains four kinases which are activated mainly by inflammatory cytokines and stress factors p38MAPKs control many aspects of cell physiology such as cell cycle regulation differentiation or skeleton remodelling Additional p38MPAKs can act as tumor promotors by enhancing metalloproteinase and VEGF expression []Wntcatenin signaling plays essential roles in caricinogenesis Activation of canonical Wnt signaling results in the inhibition of GSK3 kinase dissociation of catenin proteins and its transfer to the nucleus where it regulates multiple downstream genes like cMyc and cyclin D [] This axis was found to be associated with several oncogenic events including tumor cell proliferation migration epithelial“mesenchymal transition and invasion An abnormally active WntBcatenin pathway is observed among others in gastric [] colon [] breast [] or adrenocortical cancer [] Another signaling pathway involved in the pathogenesis of many cancer types is that induced by the Jak kinases family It includes four kinases which after activation impact of the STAT molecules causing their translocation into the nuclei [] STAT a family that gathers seven forms of proteins is associated with cancer cell development progression metastasis survival and resistance to treatment [] STAT3 and STAT5 factors seem to be the most important agents in light of cancer progression They have an impact on p53 protein which leads to a disruption in cycle control and apoptosis and induces cell proliferation by the increased cMyc and cyclin D expression By the induction of VEGF gene expression they take part in enhanced angiogenesis and induction of genes such as survivin Bcl2 BclXL conditioning overall survival of the tumor cells Moreover pSTAT3 acts to negatively regulate neutrophils NK cells effector T cells and dendritic cells while positively regulating populations of MDSCs myeloidderived suppressor cells and regulatory T cell leads to a highly immunosuppressive TME tumor microenvironment []Multiple endogenous factors are involved in carcinogenesis which affects particular stages of carcinogenesis in various mechanisms Some of them interact directly with the DNA intracellular signalling molecules others by specific cellular receptors Among them are well known growth factors EGF TGFα and TGF FGF or reactive endogenously generated oxygen molecules as well as less often described agents like ACh PPAR FABPs Btk AQPs IGF2 or exosomes The latter have only recently gained in importance and are more widely studied and discussed therefore are considered in this articleAacetylcholine Ach and its receptorsOne of the factors involved in the carcinogenesis process is acetylcholine and its receptors both muscarinic inhibitors and nicotinic Their expression has been demonstrated in numerous types of cancer cells [“] It has also been proven that these cells contain acetylcholinesterase AChE an enzyme that enables cells to produce ACh in the absence of agonists from the external environment Cancer cells also have the ability to produce autoantibodies that stimulate one of the muscarinic receptor subtypes M3 These facts prompted more extensive research to explain the exact role of ACh and its receptors in the carcinogenesis process Figure Muscarinic receptors can be divided into five subtypes each of which is involved in tumor development through a different mechanism The role of muscarinic M3 receptor is the most widely described subtype It is involved in the pathogenesis of lung colon gastric and breast cancer In the course of colon cancer it has been observed that stimulation of the M3 receptor causes phosphorylation of the Akt and ERK12 kinases which are factors that through the ability to regulate the activity of pro and antiapoptotic proteins affect the intensification of cell proliferation survival and motility In addition coexpression of the M3 receptor and endothelial growth factor receptor EGFR as well as EGFR transactivation after M3 receptor stimulation has been demonstrated [ ] Moreover the administration of EGFR inhibits acetylcholineinduced ERK12 kinase phosphorylation which is reflected in a significant decrease in colon cancer cell proliferation This indicates that acetylcholine is involved in the formation of colon cancer but for its full action it is necessary to activate the EGFR receptor Thus indicating that enriching colon cancer treatment with EGFR inhibitors may improve patient outcomes Acetylcholine is also involved in the formation of vascularlike structures in the vicinity of a developing tumor guaranteeing the supply of essential nutrients to the proliferating cancer cells This is called vascular mimicry The basis of this process is the acetylcholinedependent regulation of metastasisassociated in colon cancer1 MACC1 oncogene expression via the M3RAMPKMACC1 signaling pathway [] High activity of this oncogene is associated with an increase in cell invasiveness intensified epithelial“mesenchymal transition more frequent metastases and hence worse treatment prognosis [“] The first subtype of the M receptor M1 is also involved in the tumor development process It activates the hedgehog pathway [ ] whose physiological role is to regulate the transcription factors affecting the oncogenic activity of Gli zinc finger transcription factors [ ] Excessive stimulation causes acceleration of carcinogenesis intensification of proliferation growth and survival of cancer cells as well as angiogenesis by affecting the expression of genes such as cyclin D antiapoptotic Bcl2 or VEGF Furthermore stimulation of this pathway promotes the creation of a microenvironment conducive to tumor growth through increased expression of extracellular matrix components [] Nicotinic Oncotargetwwwoncotargetcom 0cacetylcholine receptors are part of the ligandgated ion channels and are created from two types of subunits α and Receptors that contain an alpha7 or alpha9 subunit in their structure are characterized by a high degree of permeability to calcium ions thanks to which they are involved in the course of numerous processes based on signaling involving secondary messengers Thus after activating N receptors Ca2 inflows into the cell causing the secretion of growth factors exerting paracrine action on neighboring cells Simultaneously apoptosis is inhibited enabling further proliferation Studies conducted on a lung cancer cell line after exposure to cigarette smoke have shown that nicotine and nitrosamines which have a higher affinity for nicotinic acetylcholine receptors nAChRs than nicotine bind to the alpha7 receptors activating the RasERKMAPK and the JAK2STAT3PI3K signaling pathways [ ] The effect of this interaction is increased proliferation and migration of cancer cells and thus facilitated metastasis creation Moreover by affecting Badrenergic receptors nicotine intensifies COX2 expression which through the p38 MAPK and the JNK pathways leads to an increase in VEGF production a highly proangiogenic factor In addition the high activity of cyclooxygenase affects the inactivation of the PTEN protein [ ] whose biological role is silencing PI3KAkt pathway signaling This leads to cellular signal transition and induces effects that contribute to the enhanced growth and proliferation of cancer cells as well as inhibits the process of their death Under the influence of the Akt kinase proapoptotic transcription factors are inactivated including kinase9 mTOR kinase intensifying cell proliferation is activated and VEGF is activated This indicates the involvement of this pathway in processes that facilitate cancer cell survival Another mechanism contributing to cancer development in which cyclooxygenase is involved is the complex process involving the COX2PGE2EP4 axis as a result of which metalloproteinase expression is stimulated [“] MMP9 causes proteolytic activation of TGF which begins the induction of epithelialmesenchymal transformation EMT During this transition the cells acquire features that increase their invasiveness This leads to epithelial cell phenotype changes the connections between adjacent cells and between cells and the basement Figure Carcinogenic effect of acetylcholine Ach Intracellular signaling pathways activated by ACh via M1 and M3 receptors Induction of process is illustrated by arrows inhibition by horizontal lines M1 muscarinic receptor M3 muscarinic receptor N receptor nicotinic acetylcholine receptor Akt protein kinase B ERK extracellular signalregulated kinase JAK2 Janus kinase PI3K phosphoinositide 3kinase STAT3 signal transducer and activator of transcription proteins MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cmembrane are loosened the cytoskeleton is rearranged cells lose contact inhibition thus causing excessive proliferation Furthermore the microenvironment of the growing tumor also changes promoting features that facilitate the movement of the cancer cells from the primary focus to other regions of the body Through the EMT process cells acquire greater migration capacity and thus their invasiveness increases In this cellular process MMP9 also modulates the activity of integrins and other molecules that ensure cell adhesion [] and secrete soluble factors into the bloodstream that facilitate the implantation of migrating cells in ans distal to the primary tumor focus [] Thanks to these processes COX2 enables the formation of cancer metastasesPPAR peroxisome proliferatoractivated receptorsThe family of peroxisome proliferatoractivated receptors PPARs includes isoforms of these proteins that function as transcription factors Each type of steroid receptor differs in its location and affinity for ligands [] The best known is the function of the PPARÎ form Agonists of this receptor are a therapeutic option in the treatment of patients with diabetes Current research provides evidence that stimulation of PPARÎ receptors inhibits cell proliferation vessel formation and induces apoptosis Figure [ ] These properties make PPARÎ agonists attractive candidates for anticancer drugs To achieve full transcriptional activity heterodimerization of PPARÎ with the retinoid X receptor is necessary [ ] This interaction enables the promoter to bind with the target gene and regulate its expression An example of a gene against which PPARÎ acts as a repressor is the vascular endothelial growth factor VEGF gene [] Thus angiogenesis is inhibited and this mechanism has been demonstrated in studies on human hepatocellular carcinoma HCC cells [] For this cancer PPARÎ is also involved in inhibiting cell growth and attenuating cell migration and invasiveness By upregulating KLF4 Krüppellike factor expression and inhibiting cyclin D1 expression the cell cycle is inhibited Furthermore PPARÎ negatively affects the expression of transcription factor STAT3 thereby inhibiting proliferation survival and metastasis Lowering STAT3 levels also negatively affects the angiogenesis process by inhibiting VEGF depriving cancer cells of nutrients PPARÎ activation in HHC cells also impairs their metastatic ability [] This is associated with the inhibition of the expression of metalloproteinases MMP9 and MMP13 the increased expression of their inhibitor and Ecadherin a protein conditioning cell adhesion In addition PPARÎ increases plasminogen activator inhibitor expression thus inhibiting the breakdown of the extracellular matrix and basement membrane proteins which also reduces cancer cell invasiveness PPARÎ has also been shown to inhibit esophageal cancer [] The mechanism responsible for suppressing this tumor is MAPK signaling pathway inhibition This process requires TLR4 Tolllike receptor inhibition which allows PPARÎ agonists to be administered TLR4 is a procarcinogenic protein and affects among others changes in the tumor microenvironment and the severity of angiogenesis Therefore in addition to the indirect effect on the MAPK cascade its inhibition is an important element of tumor suppression Studies on esophageal cancer cells indicate that PPARÎ stimulation leads to a decrease in PCNA proliferating cell nuclear antigen factor expression which indicates inhibition of DNA replication [] In addition PPARÎ activation intensifies the process of apoptosis as evidenced by an increase in the expression of the active form of caspase and the Bax gene and a decrease in Bcl2 expression In nonsmallcell lung cancer PPARÎ stimulation inhibits the neoplastic process by regulating PTEN protein expression [] This protein is a tumor suppressor and its activation causes the dephosphorylation and inactivation of the PIP3 second messenger As a result Akt kinase activity is inhibited leading to a decrease in NFkB expression Due to PPARÎ stimulation by eicosapentaenoic acid EPA a relationship is sought between PPARÎ and COX2 activity that uses EPA as a substrate for prostanoid production The combination of PPARÎ agonists with COX2 inhibitors has demonstrated a synergistic tumorsuppressing effect in studies on nonsmallcell lung cancer Researchers indicate significantly reduced thromboxane TXA2 synthesis using combination therapy compared with monotherapy as the main mechanism of this phenomenon [] A decrease in its concentration limits signaling in four pathways ERK p38 MAPK JAK and catenin limiting tumor growth [] Furthermore it affects the arrest of the cell cycle in the G2M phase preventing further cell division and contributing to the reduction of the expression of survivin an antiapoptotic protein [] The described mechanisms affect the induction of the apoptosis process and inhibit cell cycle progression which limits cancer development The above examples indicate that peroxisome proliferatoractivated receptors play a significant role in inhibiting the development of various types of cancer which is why agonist compounds can be considered potential anticancer drugs However further comprehensive and extensive research is needed to identify potential applications and treatment regimensBtk Bruton™s tyrosine kinaseBruton™s tyrosine kinase Btk also plays an important role in carcinogenesis Figure It is a key point in signal transduction from the BCR receptor leading to the activation of the NFkB signaling pathway [] Btk affects phospholipase CÎ2 [] which after phosphorylation causes PIP2 hydrolysis The result Oncotargetwwwoncotargetcom 0cis IP3 and DAG DAG activates protein kinase C which stimulates NFkB pathway factors Finally genes conditioning cell survival are expressed Btk also contributes to inhibiting the apoptosis process through interacting with the Akt kinase [] The signaling cascade includes active PI3K which recruits Akt to the plasma membrane Under the influence of Btk its phosphorylation occurs and thus its activation Then the active Akt returns to the cytoplasm and induces antiapoptotic pathways dependent on NFkB among others In addition Btk as a kinase belonging to the Tec family affects intercellular signaling causing changes in the tumor microenvironment In most cancer cells the developing microenvironment leads to the inhibition of immune processes which results in protection and protects defective cells from natural defense mechanisms One of the most common mechanisms of this type is the effect of cancer cells on the increased differentiation of T lymphocytes towards Th2 cells The biological role of these cells is the secretion of interleukins that start a humoral response dependent on B lymphocytes Due to the increased differentiation of T lymphocytes in this direction the number of formed Th1 lymphocytes directly destroying cells containing the abnormal genome decreases The tumor microenvironment also interferes with normal dendritic cell activity It inhibits their ability to migrate and to present the antigen to immune response cells Some types of cancer show increased CXCL12 expression [] which attracts immature dendritic cells and prevents their differentiation In addition this cytokine via the CXCR4 receptor causes a reanization of the cytoskeleton [] and increases cell motility [] Tec family tyrosine kinases have also been shown to have the ability to activate CXCR4 which determines the growth survival and migration of tumor cells []FABPs fatty acidbinding proteinsThe fatty acids supplied to the cell are involved in its metabolism Due to their lipid nature they are Figure Carcinogenic effect of peroxisome proliferatoractivated receptors PPARÎ The induction of the process is illustrated by arrows the inhibition by the horizontal line PTEN phosphatase and tensin homolog deleted on chromosome ten PIP3 phosphatidylinositol 345trisphosphate Akt protein kinase B PCNA proliferating cell nuclear antigen TXA2 thromboxane A2 ERK extracellular signalregulated kinases p38MAPK P38 mitogenactivated protein kinases JAK Janusactivated kinases KLF4 Kruppellike factor STAT3 Signal transducer and activator of transcription MMP314 metalloproteinase or PAI Plasminogen activator inhibitor TRL4 tolllike receptor MAPK mitogenactivated protein kinaseOncotargetwwwoncotargetcom 0cunable to enter the aqueous environment of the cellular cytoplasm Solubilizing molecules ie fatty acidbinding proteins FABPs enable their transport to various cellular structures and thus become involved in the processes taking place in the cells including their growth reproduction and inflammatory processes In cells characterized by intensive lipid metabolism such as the liver intestine heart brain etc high transporter expression is observed which is associated with their physiological role in the cell Depending on the type of cancer both the overexpression and a decrease in FABPs can be seen FABP isoforms are not specific to one type of cancer and each plays a different role in the carcinogenesis process Figure In liver cancer LFABP was overexpressed and its high concentration was shown to correlate positively with VEGFA [“] The causes of this phenomenon are sought in the direct interaction between these two proteins which in effect provokes the activation of the SrcFAKcdc42 and the AktmTORP70S6K4EBP1 signaling pathways Activation of the SrcFAKcdc42 pathway increases tumor cell migration and LFABP plays a key role in this process [ ] Moreover by stimulating the second mentioned signaling cascade LFABP enhances VEGFA expression facilitating angiogenesis This process is regulated by HIF1α which additionally induces blood vessel formation [] Due to the high homology in their construction FABP3 and FABP4 isoforms were assigned to the same protein subfamily In addition to their physical features it has been observed that the same factors ie HIF1α VEGF increase their coexpression The physiological role of both proteins is to inhibit excessive cell proliferation and increase apoptosis Excessive FABP3 expression leads to an increase in reactive oxygen species and a decrease in the mitochondrial membrane potential [] resulting in the ing of mitochondrial megachannels which play a key role in initiating apoptosis FABP4 activates the apoptosis process by mediating the response to lipidinduced endoplasmic reticulum stress Due to the biological functions of both proteins their deficiency may be the cause of cancer progression When examining the level of FABP3 expression in embryonic tumor cells breast cancer cells [] and FABP4 in breast ovarian prostate bladder or liver cancer cells low levels of both proteins were observed which was associated with a worse prognosis in terms of overall survival Furthermore high FABP4 levels slowed the development of hepatocellular carcinoma and thus contributed to its reduction in size [] This effect is explained by the inhibition of STAT3 phosphorylation via the RaspSTAT3 signaling pathway and the inhibition of the Snail protein an accelerator of the epithelialmesenchymal transition [] However the increased FABP3 expression in tumors of the stomach brain small and nonsmallcell lung cancer seems paradoxical it has been shown to increase tumor aggressiveness and poorer patient prognosis [ ] Thus far the molecular mechanism responsible for the oncogenic potential of FABP3 and FABP4 is unknown Further research is needed to clarify the reasons these proteins promote the neoplastic process Another subtype of fatty acid binding proteins is FABP5 It is involved in the development of breast prostate liver stomach and colon cancers FABP5 has been shown to transport saturated and unsaturated longchain fatty acids that act as PPARδ ligands [ ] The nature of the supplied fatty acids determines the oncogenic activity if unsaturated fatty acid or a suppressor action is supplied to PPARδ in the case of saturated ligands [] When the Figure Carcinogenic effect of Bruton™s tyrosine kinase Btk The induction of the process is illustrated by arrows the inhibiton by the horizontal line Btk Bruton™s tyrosine kinase CXCR2 CXC chemokine receptor type CÎ2 phospholipase CÎ2Oncotargetwwwoncotargetcom 0cFABP5PPARδ pathway is stimulated the transcription of the genes responsible for cell growth and survival increases Moreover one of the target genes stimulated by PPARδ is the FABP5 gene which increases the expression of the transport protein in question [] In prostate cancer cells by supplying long chain fatty acids FABP5 leads to PPARÎ activation [“] Stimulation of these receptors results in increased expression of vascular endothelial growth factor VEGF thereby accelerating the angiogenesis process This mechanism plays a key role in the development of castrationresistant prostate cancer [“] In the case of colon cancer FABP5 contributes to its development by reducing p21 activity Overexpression of FABP5 increases the expression of cMYC which inhibits the action of a cell cycle inhibitor After silencing FABP5 activity a significant reduction in the invasive capacity of colon cancer cells CRC is observed [] The molecular basis of this interaction is not yet known at the moment High expression of monoacylglycerol lipase MAGL responsible for the production of free fatty acids is observed in highly malignant colon cancer cells [ ] This fact combined with the physiological role of FABP5 consisting of transporting free fatty acids to the appropriate cell compartments prompts to seek the answer to the question whether FABP5 and MAGL come into functional interaction with each other and if so how does this affect CRC progression Another FABP isoform that may be involved in colon cancer development is FABP6 [“] It transports bile acids between the epithelial cells of the large intestine which are transformed into secondary metabolites such as deoxycholic acid DCA and lithocholic acid via cellular metabolism A correlation was demonstrated between increased exposure of intestinal epithelial cells to DCA and an increase in reactive oxygen species inside them which is associated with oxidative stress development and DNA damage [“] This mechanism contributes to the formation of mutations that in the event of a replication of defective cells initiate their malignancy Due to their detergent properties bile acids can cause damage to the cell membrane Compensatory mechanisms lead to an intensified inflammatory response and increased proliferation which is qualified as early tumor development Furthermore bile acids activate the muscarinic M3 receptor [“] Its stimulation induces Figure Carcinogenic effect of fatty acid binding proteins FABPs The induction of the process is illustrated by arrows the inhibition by the horizontal lineSTAT3 signal transducer and activator of transcription Src protooncogene tyrosineprotein kinase Src FAK focal adhesion kinase cdc42 cell division control protein Homolog Akt protein kinase B mTOR mammalian target of rapamycin P70S6K p70 ribosomal protein S6 kinase 4EBP1 eukaryotic translation initiation factor 4E eIF4Ebinding protein VEGFA vascular endothelial growth factor1 M3 muscarinic receptor MMPs metalloproteinases PPARÎ peroxisome proliferatoractivated receptors Î PPARδ peroxisome proliferatoractivated receptors or δOncotargetwwwoncotargetcom 0cmetalloproteinase activity and the WntBcatenin signaling pathway which in turn increases the metastatic potential as well as the proliferation and survival of colon cancer cells Desp
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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 flux 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 flux 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 signaling“cancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdaci™sheatshock 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 iscontextspecificnkg2dls 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 ofspecific 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 find 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 findingssuggest 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 scientific 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 d61345aminoimidazole4carboxamide2deoxydglucosetrifluoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulfide 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 gfpmycmicaˆ— and micaˆ— vectors containingthe coding sequences of micaˆ— or micaˆ— 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 micafirefly luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c o™callaghan university of oxfordoxford united kingdom from tocris biosciencepurification 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 mccoy™s 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoy™s 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 finger 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 confirmed 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 followingmanufacturer™s protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were confirmed by sequencing at eurofins mwgoperons luxembourg colonies were amplified and plasmidswere purified 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 amplified and dna waspurified 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 final volume of µl the dna mixture was complexed with µl 2x hbsunder constant air flow 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 micaˆ— and micaˆ—constructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbriefly dnarna were prepared in µl cacl2 25mand adjusted to a final 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 flow cytometry using accuri c6 flow 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 flow 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 manufacturer™s 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 flow cytometerwas assessed by accurate mass and retention time amrt plusfragment identification 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 confirmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturer™s 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 profilingto 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 filter 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 flight 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 inlinefilter 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 flowrate 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 lchrmsfiles were independently processed with an inhouse developedpcdl library for polar metabolites using profinder version b06agilent technologies identification of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular flux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density — cellswell ˆ¼ 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 flow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modified 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 scientificwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientific 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 manufacturer™s 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 flow 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 fluorescently 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 nkg2d“fc receptor 1299nk rd systemsand igg1“fc 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 fluorescent intensity mfi values aredisplayed in figures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and purified on directzol spincolumns zymoresearch irvine ca united states according to manufacturer™sprotocol 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 firefly 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 firefly 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 transfectionefficiency firefly 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 pcramplified 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 flow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were filtered 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 identified using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantification and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical significance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in figure 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 significance was determined by ˆ—p ˆ—ˆ—p and ˆ—ˆ—ˆ—p ˆ—ˆ—ˆ—ˆ—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 confirm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspecifically to mgat5modified nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modifiednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modification 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 specificity 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 significant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspecific 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 transfectionconfirming 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 deficiency 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 micaˆ— and micaˆ—are distinctly regulated posttranslationally and althoughmicaˆ— 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 offluorescently labeled lpha mgat5 modifications or epha mgat3 modifications on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by flow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean fluorescence 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 signifies 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
" many suggest that shared decisionmaking sdm is the most effective approach to clinical counseling itis unclear if this applies to surgical decisionmakingespecially regarding urgent highlymorbid operations in thisscoping review we identify s that address patient and surgeon preferences toward sdm in surgerymethods we used the preferred reporting items for systematic reviews and metaanalyses extension for scopingreviews prismascr to develop our protocol medline embase and cochrane databases were searched frominception through title review identified peerreviewed empirical s that addressed patientsurgeon preferences toward sdm in surgery identified s underwent full review by two independentinvestigators we addressed the following questions what is known from existing empirical evidence aboutpatients™ andor surgeons™ surgical decisionmaking preferences why might patients andor surgeons prefer sdm does acuity of intervention impact surgical decisionmaking preferences outcome measures included studymethods surgical specialty diagnosis study locationsetting typenumber of subjects acuity of intervention surgeonpatient decisionmaking preferences and factors associated with favoring sdm data was analyzed in microsoft excelresults s were identified with duplicates yielding s for title review swere included in final analysis of s discussed oncologic decisionmaking of these focused on breastcancer of s included patients included surgeons of s found surgeons favored sdm demonstrated surgeons favored surgeon guidance of s demonstrated patients favored sdm showedpatients favored surgeon guidance showed patients preferred independent decisionmaking the most commonfactors for patients favoring sdm included female gender higher education and younger age for surgeons the mostcommon factors for favoring sdm included limited evidence for a given treatment plan multiple treatment optionsand impact on patient lifestyle no s evaluated decisionmaking preferences in an emergent settings there has been limited evaluation of patient and surgeon preferences toward sdm in surgical decisionmaking generally patients and surgeons expressed preference toward sdm none of the s evaluated decisionmaking preferences in an emergent setting so assessment of the impact of acuity on decisionmaking preferences islimited extension of research to complex emergent clinical settings is neededkeywords surgery shared decision making ethics correspondence ericacarlisleuiowaedu1program in bioethics and humanities university of iowa carver college ofmedicine iowa city usa3department of surgery university of iowa hospitals and clinics iowa cityusafull 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 0cshinkunas bmc medical informatics and decision making page of over the past several decades physician paternalism hasbeen systematically rejected and respect for patient autonomy has emerged as a leading ethical priority in clinical counseling shared decisionmaking sdm aprocess by which physicians and patients actively worktogether to integrate care plans that are responsive topatient goals and values has been advocated as a clinicalcounseling approach that promotes patient autonomy byencouraging patients to participate in clinical decisionmaking [“] along with its presumed promotion ofpatient autonomy data suggesting that sdm reduceshealth care costs and improves quality of care have ledto relatively widespread incorporation of sdm intohealth policy despite this implicit acceptance ofsdm relatively limited data exist regarding patient orphysician preferences toward sdm such data seem tobe especially lacking in surgical decisionmakingby supporting patient autonomy sdm places somelimits on the extent to which a physician™s influenceguides a patient™s decisions some ethicists have arguedthat such prioritization of patient autonomy is criticallyimportant and that even subtle attempts by a physicianto sway a patient toward a particular decision violates respect for patient autonomy however others have argued that if attempts to promote patient autonomy aretoo strong or rigid the emphasis on selfdeterminationmay be inconsistent with patients™ wishes for more professional guidance [ ] in fact there is an emergingbody of literature that suggests that patients may prefermore physician guidance during medical decision making [ “] these findings prompt one to questionwhether autonomyheavy approaches to sdm in clinicalcounseling are always consistent with patient preferencesor whether patients would at least sometimes prefer aless autonomous and more guided approach to clinicalcounselingwith respect to the physician™s perspective it is important to note that studies have shown physicians to besomewhat reluctant to incorporate sdm into clinicalpractice one reason for this may be a sense thatwhen a physician overly prioritizes patient autonomythere is lessening of the physician™s role such that the fiduciary nature of the patientphysician relationship isundermined prioritization of patient autonomy and integration of sdm into clinical counseling has left somephysicians feeling that their role has become one ofmerely offering patients the information necessary tomake their own œinformed decisions rather than trulyengaging in a fiduciary relationship with the patient this is illustrated in a recent narrative that describes anencounter in which a physician reviewed all options fortreatment of nonischemic cardiomyopathy with her patient but was stopped by the patient before she couldmake a recommendation with the request that the patient be allowed time to independently reflect and makea decision that was best for him in the physician™s reflection on the encounter she notes œsince the decisionwas his it was no longer mine i had informed him buthad i been his doctor perhaps such efforts to assure patient autonomy and sdm limit the role of thephysician in patient counseling these types of reportscall for further investigation so we can better understandphysician preferences toward shared decision makingconcerns about the appropriateness of sdm may beparticularly pronounced in surgical decision making giventhe often dramatic and irreversible outcomes associatedwith surgery these concerns may further escalate whenconsidering emergent highly complex operations that areassociated with a high risk of mortality or morbidity in aninitial effortto better understand preferences towardsdm in surgical decision making we reviewed the literature regarding parent and surgeon preferences towardsdm in pediatric surgery we found that there wasmarkedly limited data available of the existing sthe predominant focus was on parent preferences towarddecision making in elective nonurgent procedures therewas limited data regarding surgeon preferences and virtually no discussion of preferences for decision making inmore urgent settings the purpose of this review is to gain a more thorough understanding of patient and surgeon preferences toward sdmin adult surgery we chose to conduct a scoping review because there is limited published data on patient and surgeondecision making preferences particularly when surgery isconsidered urgent or emergent scoping reviews are a valuable methodology because they allow for the mapping of important concepts and research gaps in a defined area ofstudy by comprehensively identifying reviewing and summarizing the existing information from the literature specific research questions addressed in our scoping reviewincluded what is known from existing empirical evidence about patients™ andor surgeons™ surgical decisionmaking preferences why might patients andor surgeonsprefer sdm does acuity of intervention impact surgicaldecisionmaking preferencesmethodsprotocol designour scoping review protocol follows arksey and o™malley™s methodological framework as well as the preferred reporting items for systematic reviews andmetaanalyses extension for scoping reviews prismascr this protocol has not been registeredidentifying relevant studiesafter ascertaining our research questions we worked inconjunction with an experienced medicallibrarian to 0cshinkunas bmc medical informatics and decision making page of identify relevant studies we followed the preferredreporting items for systematic reviews and metaanalyses prisma guidelines for reporting the identified screened eligible and included studies fig after drafting refining and finalizing our search strategies we searched three bibliographic databases from inception through november medline embaseand cochrane databases the final search strategies forall three databases are outlined in additional file thefinal search results were imported into endnote versionx91 and yielded sstudy designeligibility criteriainclusion and exclusion criteria were defined a priorireview was limited to english language no translatorsavailable peerreviewed published literature only empirical studies were included review was limited todecision making preferences of surgeons andor adultpatients decision making preferences were loosely defined and included œpreferred role œperceived role œexpectationsœdesires and œsatisfaction with actualdecisionmaking role s in the following categories were excluded reviews letters to the editor editorialssuggested models of care patient educationhandouts decision making tools animal studies and s related to pediatric surgery in addition we excluded s without accessible full textliterature reviewafter duplicates were removed by the primary authorlas we were left with s to screen twoof the authors las and emc independently reviewedall titles and s and jointly decided to exclude s based on the eligibility criteria theremaining s were selected for full text reviewfig flow diagram of study selection 0cshinkunas bmc medical informatics and decision making page of following full text review additional s were excluded because they either did not pertain to an adultsurgical population or to decisionmaking preferences inthe surgery setting disagreements were resolved by discussion between the two authorssubjects gender acuity ofsurgeryothercharting the datafor each of the included s two of the authorslas and cjk independently ed the followingoutcome measures study methods quantitativequalitativemixed methods surgical specialty cancer diagnosisyesnounclear study location usnonus study setting inpatientoutpatient type of subject patientsurgeon number oftheintervention electiveurgentemergentunclear surgicaldecision to be made surgery v nonoperative managementchoice among different surgical proceduresdecision on timing ofsurgeonpatientdecision making preferences shared decision makingsurgeon guided decision making independent decisionmaking and surgeonpatient factors associated with favoring sdmacuity ofthe intervention was defined as followsemergent immediate need for surgery to preserve lifeurgentsurgery is required within the next days orweeks and elective surgery is not required notablycancer resections were considered urgent however subsequentreconstruction was considered elective iebreast cancer resection with subsequent reconstructionthe control preferences scale which is a fivepointmeasure used to gauge preferred involvement in medicaldecision making was adapted to define surgeonpatient preferences as follows shared decision makingsdm the patient and surgeon prefer to make the decision regarding surgery together surgeon guided decisionmaking sg the preference is for the surgeon to guidedecision making either entirely or in part while the patient takes a more œpassive role independent decisionmaking idm the preference is for the patient to take amore œactive role in decision making either partly orentirely independent from the surgeonthe data ion form was a modified version ofthe one we used for a literature review we conducted ondecision making preferences in the pediatric surgical setting discordant opinions were discussed at weeklymeetings the third author emc was available to mediate if consensus could not be reached data was analyzed in microsoft excel resultssummarizing collating and reporting the resultssummarizing the results s were identified medline n embase n cochrane n duplicateswere removed and s underwent title review seventyfour s were included in thefinal analysis because they specifically addressed existingempirical evidence about patient andor surgeon decision making preferences toward sdm in adult surgerysetting table provides a summary of allincludedscollating and reporting the resultstable provides frequencies for the characteristics of allincluded s over half of the s were quantitative n and performed outside of the us n sixtyseven included outpatient surgeries fourteen surgical subspecialties were representedwith the most s originating from surgical oncology n general surgery n orthopedic surgery n and urology n fifty s discussed decision making for patients with cancer and of these focused onbreast cancer most s assessed a choice betweenoperative and nonoperative management n or an option among different surgical procedures n sixtyeight of the s included patients ofthese demonstrated that patients preferredsdm showed that patients favored a surgeonguided approach and revealed a patient preference for independent decision making the most common factors for patients favoring sdm included femalegender higher education and younger ageonly of the s assessed surgeons™ preferences of these found that surgeons preferredsdm while demonstrated that surgeons favoreda more surgeonguided decisionmaking approach thefactors most commonly listed for surgeons favoringsdm included limited evidence for a given treatmentplan multiple treatment options and impact on patientlifestylenone of the s evaluated patient decisionmakingpreferences in an emergent setting out of the sthat assessed patient decisionmaking preferences in theelective surgery setting preferred sdm threeout of four of the s assessing surgeon decision making preferences in the elective surgery settingreported that surgeons preferred sdm in six out of nine of the s surgeons also preferred sdm in theurgent surgery setting in s patients were fairlysplit on their decisionmaking preference when it cameto urgent surgeries with desiring sdm and favoring a more surgeonguided approachonly s looked at both patient and surgeon decision making preferences in a little over half ofthese s n there was discordance between patient and surgeon decision making preferences 0cshinkunas bmc medical informatics and decision making page of table characteristics of included sacuity of andthedate ofpublicationinterventionstudy populationmajor findings related to decisionmaking dm preferencesalmyroudi ananian andersen asghari ashraf avis ballinger breast cancerpatients breast cancerpatientsurgentelective breast cancersurvivorsurgent hospitalized patients on surgical wardsunclear patients undergoingeither immediate ordelayed breastreconstructionelective hernia repair patientselective breast cancerpatientsurgentbeaver colorectal cancerpatientsurgentbeaver belue health professionalscaring for colorectalcancer patients weresurgeons cardiologists making adecision about surgery patients with coronaryartery diseaseblumenthalbarby left ventricular assistdevice patients andcandidatesburton older breast cancerpatientsbutow patient advocates breast cancersurgeonsurgenturgenturgenturgenturgent preferred a passive role acollaborative role46 an active role of women choosing breastreconstruction œdecided with surgeon of these patients were satisfied withthe information receivedon average reported being œveryinvolved i made all the decisions myself were content with dm roleœstrongly desire to receive informationand participate in decisionmaking were in the œinformedconsumeristgroup when it came to actual dm of these patients were satisfied with theinformation receivedœexpectations of participation can besummarized as ˜being told™ and ˜going into get it fixed™ œfelt their healthcare professionalshad surgical preferences for thembelieved that clinical issues determinedthese preferences but still knew thechoice was theirsœwanted to be well informed andinvolved in the consultation process butdid not necessarily want to use theinformation they received to makedecisionsœshared decision making was favored byhealth professionalsphysicians œprefer patients whoactively participate in the decisionpatients œprefer the physician tomake the decision sdm œprefer to make the decision on theirownœdeferred heavily to clinicians preferred œpatientcentred œdoctorcentred sdm of surgeons and of patientadvocates preferred sdm breast cancer survivors urgentcampesino cohen patients with localizedprostate cancerurgentcorriere patients undergoingelectivespanishspeaking latinas preferredœphysician treatment recommendationsenglishspeaking latinas and africanamericans preferred sdmmost viewed the surgeonguided approach as œappropriate and welcome preferred œchoosing together withdm themerelated tomajorfindingsaptsgsurg“factors associated with favoringsdmyounger age higher educationsdm “type of procedureidm“younger age level of educationincomesdm “female level of educationidmsg““sdm “sg““sdmyounger patient agesgsdm““sgsgidmsdm sdmsdmsgsg““englishspeakingsdm “multiple treatment options type 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencesfactors associated with favoringsdmdm themerelated tomajorfindingsaptsurg elective vascularprocedures prostate cancersurvivors hand surgeons patients with triggerfinger breast cancerpatientsurgentelectiveurgentcuypers doring durifbruckert gainer the provider preferred œhaving theprovider choose for them preferred a collaborative role an active role a passive roleof proceduresdm “higher education younger agehigher sespatients œpreferred to decide forthemselves surgeons preferred sdmidmsdmwanted to participate in decisions butœperceived sdm as an obligation becauseit did not seem to fit with their idea of aproper doctorpatient relationshipsg“trust in surgeon support fromfamily written information fromsurgeon frail and olderpatients care teammembers includessurgeonsunclearboth patients and care team membersœsupported a formal approach to sdmsdm sdmghane general surgerypatientsgolden clinicians weresurgeonsgong patients with carpaltunnel syndromeelectiveurgentœpreferred relatively high levels ofdecisional control on averagem out of sd most felt that they practiced sdm eventhough they did not tend to distinctlyprompt patient dm preferencesidm“male good health high healthliteracy“sdmelective preferred sdmsdm “hack breast cancerpatientshageman hand surgeons patients with carpaltunnel syndromehawley breast cancerpatientsheggland hausken health professionalsfrom surgical wards patients who underwentsurgical treatmenturgentelectiveurgentelectiveheggland hausken surgical patients surgeonselectiveurgentheggland [ ]henderson shum physicians workingin surgical wardsunclear surgical and medicalpatientselectiveurgent preferred a collaborative role an active role a passive rolesurgeons preferred œpatient andprovider make a shared decisionpatients preferred that œthe patientdecidesactual dm role sdm œpatientbased œsurgeonbased preferreddm role content with level of dminvolvementhealth professionals majority preferred aœshared or œinformed model patientsabout half preferred a œshared orœinformed model and the other halfpreferred a œpaternalistic modelsurgeons the majority preferred anœinformed model ¦ patient is giveninformation and left to make thedecision patients preferred aœpaternalistic model and preferredsharedphysicians on average rated decisionmaking control a which means thatœphysicians were not reluctant to involvepatients in decisionmaking processeswhere active role shared and passive “ the mean dm value for thesevere scenario was moderatescenario was mild scenario was history of surgical procedureimportance of family memberopinions having privateinsuranceage nonwidowed longerduration postopsdm “idmsdm“sdmidmsdmfemalesdmsgsgidm“sdmsdm “younger age noncriticalcondition 0cdm themerelated tomajorfindingsaptsdm “surgfactors associated with favoringsdmfemale higher educationsgsg“““female no stomasdmmore years in practice morecomfort in pulmonary nodulemanagementcollege degree higher selfefficacysdm “sdm “sg“sdm “œfemales indicated that they would like tohave more input in the decisionmakingprocess than the males v onthe controlled preferences scaleœguidance by the clinician was identifiedas most important œactive role of patientin treatment decision making regardedas less important preferred a passive role sdm an active roleperceived role œshare decisionsequally with the patient œallowthe patient to decide œdecide forthemselves after considering the patient™sopinionœmost patients wanted to decide on theirtreatment options together with theirphysician preferred sdm preferred tomake the decision œwith physician inputœ wanted their surgeons to make arecommendation and when given followed the recommended treatmentplan preferred a collaborative role inpatients in twosurgical unitsunclear lung cancer patientsurgenthenderson hopmans hou colorectal cancerpatientsiaccarino clinician members ofthe american thoracicsocietyurgenturgentihrig prostate cancerpatientsjanz breast cancerpatients newly diagnosedbreast cancer patientsurgenturgenturgent breast cancerpatientsurgentjohnson keating keating lally shinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences lung or colorectalcancer patientsurgent œpatient controlled sdm œphysician controlledsdm “married better prediagnosishealth status caucasian strongevidence for procedure breast cancer patients urgentœwomen™s lack of sharing theirpreferences with their surgeons and thesurgeons™ lack of making treatmentrecommendations resulted in what wasmore likely informed than shared decisionmaking preferred sdm preferred œthechoice to be their own preferred œtodelegate the decisionactual role sdm madedecision with œsurgeon input weresatisfied with dm levelactual dm œjoint patientdoctor decision œdoctor advocated œpatient asked preferred dm contentwith level of dm involvementthe surgical group showed a morepassive role in both their preferred andactual dm roleœsurgeon™s recommendationand fear of dying from cancer played themost important role in dmin surgery preferred a œdirectivecommunication style a œnondirective communication styleidm“sdm “younger agesdm “sdm “femalesgsg““sdmsglam breast cancerpatientslantz breast cancerpatientsurgenturgentlarsson patients scheduledfor invasive surgeryelectiveurgenturgentlee patients with earlygastric cancermarkovic martinez newly diagnosedgynecologic cancerpatients newly diagnosedbreast cancer patientsurgent 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences surgeonsunclearmcguire mendick breast cancer patients surgeonsurgentmeredith surgical patients surgeonsunclear older breast cancerpatientsurgent patients withirritable bowel diseaseelective breast cancer patients urgentman morishige moumjid nam patients with carpaltunnel syndromeelectiveomar op dendries consecutive patientsbeing seen in a multidisciplinary stone clinicelective liver transplantcandidates and recipientsurgentorsino end stage renaldisease patientselectiveœmany physicians saw their role as anexpert who educates the patient butretains control over the decisionmakingprocessothers took a more collaborativeapproach encouraging patients toassume decisional prioritysurgeons œmade most decisions forpatients patients œgenerally lacked trustin their own decisions and usually soughtsurgeons™ guidancepatients œmajority agreed that thesurgeon should supply them with the˜pros™ and ˜cons™ of all measures toaddress the problem and it was for themultimately to decide what was right forthem surgeons œnot enthusiastic at theprospect of devoting more time todiscussing surgical alternatives risks andcomplications and outlook indicators fortheir patients benefitin surgery preferred sdm aœdoctorcentered approach œ aœpatientcentered approach œthought having a physician involvethem in the decisions concerning theirtreatment was very importantœmost were satisfied with the informationgiven and the possibility of participatingto the treatment decisionmakingprocessœi prefer that my doctor and i shareresponsibility œœi prefer that mydoctor makes the final decision aboutwhich treatment will be used butseriously considers my opinion œwould rely on the physician™srecommendationœ wished to be involved in makingthe decision to accept or not accept aliver for transplantation preferred œequal responsibility an œautonomous role adecision driven by the health care teamdm themerelated tomajorfindingsapt“surgsdmsgfactors associated with favoringsdmmultiple treatment optionsincreased risk impact ofprocedure on patient lifestylemoral contentsgsgsdm sgpatients strong evidence forprocedure surgeons multipletreatment options impact ofprocedure on patient lifestylesdm “older agecomorbidities surgical historyuse of biologics treated at anacademic hospital being marriedsdm “sdm “sdm “sg“sdm “sdm “younger agepieterse ramfelt rectal cancer patients surgical oncologistsurgentthe majority of patients and clinicianspreferred sdmsdm sdmpatients female highereducation rectal or colon cancerpatientsurgent of rectal cancer patients ofcolon cancer patients preferred acollaborative rolesdm “younger ageratsep patients with lumbardisc herniationelective preferred sdmsalkeld rectal or coloncancer patientsurgent preferred a surgeonguided approach sdm a more independent dm rolesdm “sg“desire for more disease specificinformationfemale younger age history ofradiation 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencessantema patients with eitherabdominal aorticaneurysm or peripheralarterial occlusive diseaseelective preferred sdmseror young breast cancerpatientsurgentsidana young prostatecancer patientsurgentsnijders stiggelbout kiebert gi surgeonsurgent cancer patients surgical patientsunclearpreferred a more passive approach preferred œfully passive and preferred fairly passive preferred sdm an œinformeddecision made by myself based oninformation a passive roleœmost patients were offered only onetreatment option and little sdm wasseenœthe physician should make the decisionsbut strongly consider my opinion wasselected most frequentlyfactors associated with favoringsdmtrust in doctor doctor has aclear communication styledoctor listens enough time forconsultationhigher education type ofproceduredm themerelated tomajorfindingsaptsdm “surgsg“sdm ““sgsg“younger age femalesung patients with pelvicfloor disordertyler ellis newly diagnosedrectal cancer patientsuldry patients undergoingelective gi surgeryvogel breast cancerpatientswang breast cancerpatientsurgenturgent spine clinic patientselectiveweiner essis wilson patients undergoingmajor thoracicabdominal operationsurgentelectivewoltz patients withdisplaced midshaftclavicular fractureelectiveurgent preferred a collaborative role an active role a passive rolesdm “ of total mesorectal excision patientsand of local excision patientspreferred sdmsdm “higher education younger ageelective preferred an active roleidmyounger age male level ofeducationhigher anxiety scores multipletreatment options preferred a passive role anactive role sdm preferred a collaborative role a passive role an active roleœthe majority of patients felt that thephysician rather than the patient shouldmake the basic treatment decision preferred a œpatientdriven role sdm a œsurgeondriven rolesg“sdm “sgidm““ preferred sdm œautonomousrole a passive rolesdm “ ovarian cancerpatientsziebland adecision making preference dm decision making sg surgeonguided sdm shared decision making idm independent decision makingdx diagnosis pt patient surg surgeonœpreferred their medical team to decideon their behalf or œ˜going along with™their doctor™s recommendationurgentsg“out of these s three focused on elective surgeriesin orthopedics one on urgent surgeries in cardiac surgery one on both elective and urgent surgeries in general surgery and one was unclear on the acuity of theinvention but occurred in general surgerydiscussionshared decision making has been highlighted as a desirable approach to clinical counseling however it isunclear if this applies to surgical decision making particularly when considering surgical counseling in settingsof emergent complex highlymorbid operations inour scoping review of the adult surgical literature wefound relatively few studies that address patient and surgeon preferences toward sdm in surgery we found thata large proportion of existing s on preferences toward sdm address elective outpatient procedureswhile patients did seem to prefer sdm in these controlled settings it is possible that patients and surgeonsmay prefer more surgeon guidance when discussingemergent complex operations that have a high risk ofmorbidity or mortality further studies that specifically 0cshinkunas bmc medical informatics and decision making page of table frequencies for characteristics of all included sn variablesurgical specialtyastudies noncologygeneral surgeryorthopedicsurologygynecologycolorectalthoraciccardiacplastic surgerytransplantationvascularneurosurgeryentotolaryngologyophthalmologycancer diagnosisyesnounclearstudy methodsqualitativequantitativemixed methodsstudy locationusnonusstudy settinginpatientoutpatientbothtype of subjectspatients onlysurgeons onlybo
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"unrestricted use distribution and reproduction in any medium provided the original work is properly citedPurpose The present study was aimed at determining the serum levels of actinin4 ACTN4 in cervical cancer CC andinvestigating the diagnostic and prognostic value of serum ACTN4 in CC Materials and Methods We included CC patients cervical intraepithelial neoplasia CIN patients and healthy women Serum ACTN4 levels were assessed using an ELISAmethod A receiver operating characteristic ROC curve was performed to evaluate the diagnostic value of serum ACTN4 Thesurvival curves were used to display the overall survival distributions Results Serum ACTN4 levels in CC patients were ± pgmL which is significantly higher than those in CIN patients ± pgmL P and those in healthycontrols ± pgmL P The ROC analysis demonstrated that the area under the curve AUC of ACTN4 was 95CI “ with sensitivity of and specificity of Serum ACTN4 levels were associated with theFIGO stage lymph node metastasis and lymphovascular space invasion of CC all P The survival curve suggested thathigh serum ACTN4 levels were related to poor prognosis Conclusion Our findings suggest that serum ACTN4 levels may bevaluable diagnostic and prognostic biomarkers for CC IntroductionCervical cancer CC is the second most common femalemalignancy globally and it is the most common femalemalignancy in developing countries which has high morbidity and mortality rates [] In recent years the incidence ofCC has increased greatly in young women under the age of [] Despite great advances in surgical and adjuvant therapy the overall survival of CC patients especially that ofadvanced patients is still very poor [] At present a Papsmear combined with an HPV test has been used for the earlyscreening of cervicalthe screeningmethods are invasive and costly leading to lower screeningcoverage in China [] Previous studies have reported thatthe human papillomavirus HPV screening results have arelatively high falsepositive rate and a relatively low specificity [ ] In addition the results of TCT interpretation byfilmreading doctors are uneven which might cause somelesions Howevermisleadingness in the choices of prevention measures andtreatment for CC [] Noteworthily when applying the sametreatment plan to patients with similar pathological types theefficacy and prognosis are quite diï¬erent Therefore it is necessary to identify new biomarkers directly related to the progression and prognosis of CCAlphaactinins ACTNs are actinbinding proteins inthe spectrin gene superfamily [] which are known to becrosslinked with filamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1“which are present in humans and other mammals [“]ACTN4 is encoded by the ACTN4 gene and is widelyexpressed in many tissues especially in glomerular podocytes[] ACTN4 has an actinbinding domain at the Nterminus and ACTN4 monomers can form a homodimer throughreverse binding forming a dumbbellshaped structure []As an actinbinding protein ACTN4 is closely related to 0cDisease Markersenhancing cell viability and tumor invasion and metastasis[] Recent researches have reported that the expression ofACTN4 is significantly elevated in multiple cancers including breast cancer [] pancreatic cancer [] ovarian cancer[] and lung cancer [] In addition the ACTN4 levels aremarkedly associated with the poor prognosis of lung cancer[ ] thyroid cancer [] and salivary gland carcinoma[] An [] have found that the expression level ofACTN4 in human cervical tumors is dramatically higherthan that in normal cervical tissues Their finding demonstratedepithelialtomesenchymal transition and tumorigenesis by regulatingSnail expression and the Akt pathway in CC [] Thereforethe expression of ACTN4 in cervical tissues may be used inthe clinical diagnosis and prognosis prediction of CCthat ACTN4promotestheHowever up to now the significance of the serumACTN4 levels in CC has not been evaluated Hence in thecurrent study the serum levels of ACTN4 in patients withCC were measured In addition we estimated the potentialdiagnostic and prognostic value of serum ACTN4 expressionin CC Materials and Methods Study Population A retrospective study was designed toevaluate serum actinin4 as a biomarker for CC Between July and June newly diagnosed female CC patientsand newly diagnosed female cervical intraepithelial neoplasia CIN patients who received treatment at Huai™anMaternal and Child Health Care Hospital Huai™an JiangsuChina were recruited The diagnoses of all patients were verified by the histopathological examination The patients withother types of tumor or autoimmune atherosclerotic andhematologic diseases were excluded The mean age of CCpatients was years with a range of years Meanwhile healthy women with no evidence of neoplasmsand other serious diseases were enrolled from the physicalexamination center in the same hospital There was no significant diï¬erence in age among the CC CIN and healthy control groups This study was consistent with the Helsinkideclaration and was authorized by the Ethics Committee ofHuai™an Maternal and Child Health Care Hospital approvalnumber H20130504 All participantssigned writteninformed consent Clinicopathologic Feature Collection and FollowUp Byreviewing the medical records we collected the clinicopathologic characteristics of the patients including age at diagnosis pathological type FIGO stage tumor diï¬erentiationpelvic lymph node metastasis tumor size and lymphovascular space invasion The CC patients were classified based onthe revised FIGO staging system for CC in The tumorsize was the maximum tumor diameter determined by agynecologic oncologist during pelvic examination Thepatients in stage 1A1 received hysterectomy the patients instages IB1 and IIB received radical hysterectomy and pelviclymph node dissection the patients with ‰¥stage IIB receivedradiotherapy or radiotherapy combined with chemotherapyA regular telephone followup was conducted after treatmentto obtain the overall survival OS time of CC patients andthe OS was defined as the time from diagnosis to death orthe last followup The followup was in accordance withthe FIGO guidelines Blood Sample Collection and Detection of Serum Actinin and SCCA A mL peripheral blood sample from eachpatient was collected before receiving any treatment Afterstanding at room temperature for minutes the blood samples were centrifugated at gmin for min and then°the supernatant was stored at ˆ’C until further usageThe serum actinin4 concentration was measured by a quantitativeELISAmethod Uscn Life Science Inc Wuhan China The levelsof SCCA in serum were determined using an ELISA kitRD Systems Minneapolis MN The detection of all samples was strictly in accordance with the instructions providedby the manufacturer and was performed in duplicatesenzymelinked immunosorbentassay Statistical Analysis All statistical analyses were conducted by using SPSS and GraphPad Prism The continuous data following normal distribution were expressed asthe mean ± standard deviation°SDž A ttest was used tocompare serum ACTN4 levels between the two subgroupsof each clinicopathological parameters and the serumACTN4 levels of CC patients CIN patients and healthy controls were compared by the SNKq test Receiver operatingcharacteristic ROC curves were performed to assess thediagnostic value of serum ACTN4 levels for diï¬erentiatingCC patients from CIN patients and healthy controls TheKaplanMeier method and logrank test were used to plotsurvival curves The Cox proportional hazards models in univariate and multivariate analyses were used for evaluating theprognostic value of serum ACTN4 expression A twotailed Pvalue was considered to be statistically significant Results Serum ACTN4 Levels Are Higher in Patients with CCSerum concentrations of ACTN4 were detected to rangefrom to pgmL with a mean ±SD of ± pgmL for CC patientsto range from to ngmL with a mean ±SD of ± pgmL forCIN patients and to range from to ngmL witha mean ±SD of ± pgmL for healthy controlsSerum ACTN4 levels in CC patients were significantly higherthan those in CIN patients and healthy controls P However no significant diï¬erence in serum ACTN4 wasfound between CIN patientscontrolsP as shown in Figure and healthy The Diagnostic Value of Serum ACTN4 Levels for CC Wenext used ROC curve analysis to estimate the diagnostic valueof serum ACTN4 expression for CC The ROC curve showedthat the serum levels of ACTN4 were robust for discriminating CC patients from benign and healthy control subjectswith an area under the curve AUC value of 95CI “ as demonstrated in Figure index we usedAccordingto maximum Youden™s 0cDisease MarkersŽŽlymph node metastasis were the independent prognostic factors for CC all P Table Lmgp NTCAnsCCCINCON DiscussionCervical cancer is a heterogeneous disease with complicatedetiology Genetic and environmental factors play a crucialrole in the pathogenesis of CC [] Although the diagnosisand prognosis of CC have improved greatly over the pastfew decades it is necessary to improve early detection andscreening methods to determine additional promising circulating biomarkers for better patient selection and more personalized treatments [] As far as we know this studyrepresented the first eï¬ort to evaluate the serum expressionof ACTN4 as a new biomarker for CCAs an actinbinding protein ACTN4 can participate inregulating cell migration invasion and metastasis via regulating the actin filament flexibility at the leading edge ofinvading cancer cells [ ] ACTN4overexpressing cancercells have the potential to metastasize because the overexpression of ACTN4 protein in cancer cells can stimulate thedynamic reconstruction of the actin cytoskeleton [] Upto now numerous studies have reported the associationbetween ACTN4 and multiple cancers Okamoto []observed that ACTN4 is expressed in smallcell lung cancerNSCLC and it had a significant correlation with invasionand distant metastasis Additionally ACTN4 was reportedto be a potential predictive biomarker for the efficacy of adjuvant chemotherapy in patients with NSCLC [] Watabe [] revealed that the copy number increase of ACTN4is a novel indicator for poor overall survival of patients withsalivary gland carcinoma and the copy number variationwould aï¬ect the expression of protein A recent study demonstrated that serum ACTN4 levels were dramatically elevated in patients with breast cancer when compared tohealthy controls and serum ACTN4 may be an eï¬ective clinical indicator for diagnosing or predicting the clinical outcomes of breast cancer patients [] In addition ACTN4was proven to be associated with the pathogenesis of CCAn [] proposed a novel mechanism for epithelialtomesenchymal transition and tumorigenesis in CC whichcould be induced by ACTN4 through regulating Snail expression and βcatenin stabilization Hence it is significant toinvestigate the role of serum ACTN4 in CCIn the current study we observed that serum levels ofACTN4 in CC patients were statistically higher than thosein CIN patients and those in healthy controls Howeverserum ACTN4 levels were not significantly diï¬erent betweenthe CIN group and the control group It was shown thatserum ACTN4 expression could strongly diï¬erentiate CCpatients from CIN patients and healthy controls The ROCanalysis demonstrated that the AUC of ACTN4 was and at the optimal cutoï¬ of pgmL the sensitivity andspecificity were respectively and suggestingthat serum ACTN4 might be a potential diagnostic biomarker for CC In a recent study which included Chinesewomen Hu [] reported that the sensitivity and specificity of HPV screening in the diagnosis of CC were and The sensitivity of the HPV test was a litter higherFigure The serum ACTN4 levels in CC patients CIN patientsand healthy controls ˆ—P pgmL as the cutoï¬ value and the sensitivity and specificity were and respectively Association between Serum ACTN4 Levels andClinicopathological Parameters of CC Patients We furtherinvestigated the correlations between serum levels of ACTN4and clinical pathological data of CC patients and theresults are demonstrated in Table We observed that serumACTN4 levels were related to the FIGO stage lymph nodemetastasis and lymphovascular space invasion all P Nevertheless no significant association was found betweenserum ACTN4 levels and age pathological type diï¬erentiation degree and tumor size in CC patients all P Survival Analysis of Serum ACTN4 Levels in CC Duringthe followup period nine CC patients were lost and thefollowed up rate is Finally the prognostic value ofserum ACTN4 was assessed in patients The patients werefollowed up to December The range of followup timewas to months with the median time of months andmean time of months According to the median serumlevels of ACTN4 in CC patients pgmL the CCpatients were divided into the high ACTN4 level group pgmL N and low ACTN4 level group‰¥ pgmL N The estimated 5year OS of patientswith high serum ACTN4 levels and low serum ACTN4 levelswere and respectively The KaplanMeier survival curve and logrank test indicated that CC patients withhigh serum ACTN4 levels had a worse prognosis than thosewith low serum ACTN4 levels P Figure Univariate Cox regression analyses showed that theserum ACTN4 levels P FIGO stage P diï¬erentiation degree P lymph node metastasisP and lymphovascular space invasion P had significant prognostic value for OS Multivariate analysiswas further performed to evaluate the prognostic value ofserum ACTN4 as an independent factor for CC All the statistically significant factors from univariate analyses wereincluded and the results indicated that the FIGO stage and 0cDisease MarkersytivitisneS ˆ’ specificityFigure ROC curve analysis assessed the diagnostic performance of serum ACTN4 in CC The AUC was P Table Serum ACTN4 levels in CC patients according toclinicopathological parametersParametersAge years‰¤Pathological typeSquamous cell carcinomaAdenocarcinomaFIGO stageIA1IB1‰¥IB2Diï¬erentiationWell and moderatelydiï¬erentiatedPoorly diï¬erentiatedLymph node involvementNegativePositiveTumor size‰¤Lymphovascular space invasionNegativePositiveN ACTN4pgmLP ± ± ± ± ± ± ± ± ± ± ± ± ± ± than that of serum ACTN4 detection though the specificityof serum ACTN4 detection was well above that of the HPVtest Hence comparing with the HPV test in diagnosingCC detecting serum ACTN4 has some advantages Furthermore serum ACTN4 levels have been indicated to be a greatbiomarker for diagnosing multiple cancers Fang [] intheir study reported that serum ACTN4 was a promisingindicator for diagnosing breast cancer with the AUC of Wang [] used ACTN4 expression in peripheralblood to diï¬erentiate NSCLC patients from healthy individuals in two groups of participants and they obtained bothsatisfactory eï¬ects Furthermore we investigated the correlation between serum ACTN4 and clinical characteristics ofCC patients The serum ACTN4 levels were significantlyassociated with the FIGO stage lymph node metastasis andlymphovascular space invasion of CC which suggests thatACTN4 could contribute to the development invasion andmetastasis of CC In addition our results indicated that highACTN4 levels were associated with the poor survival of CCpatients In the multivariate analysis although ACTN4 levelsdid not reach the statistical significance it still seems to beable to influence the OSHowever several limitations in the present study should betaken into consideration First the sample size was relativelysmall which was likely to reduce the statistical power of ourresults Second we only explored the relationship betweenserum ACTN4 and OS and other prognostic indicators werenot examined due to the incomplete data which needs to beimproved in the future Third this study was a primary studyto determine the clinical significance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the specific molecular mechanisms remain unclear Hence further experimentsshould be conducted to elucidate the mechanismsIn conclusion our study showed that serum ACTN4levels were increased in CC patients and were related to the 0cDisease Markers lavivrus muCLog rank P Overall survival monthsLow ACTN4 groupHigh ACTN4 groupFigure KaplanMeier curve compared OS of CC patients with high serum ACTN4 levels versus those with low serum ACTN4 levelsTable Univariate and multivariate Cox regression analysis of OS in CC patientsUnivariate CIVariablesAge vs ‰¤ yearsPathological type squamous cell carcinoma vs adenocarcinomaFIGO stage ‰¥IB2 vs IA1IB1Diï¬erentiation poorly diï¬erentiated vs well and moderately diï¬erentiated “Lymph node involvement positive vs negativeTumor size vs ‰¤ cmLymphovascular space invasion positive vs negativeSerum ACTN4 levels high vs low levelsHR “ “ “ “ “ “ “ “ “ “ “ “P”””Multivariate CIPHRFIGO stage lymph node metastasis and lymphovascularspace invasion of CC patients In addition serum levels ofACTN4 have great diagnostic and prognostic value in CCNevertheless further studies with a larger sample size shouldbe carried out to confirm our resultsAcknowledgmentsWe thank all the patients and blood donors who participatedin our study This study was funded by grants from the Science and Technology Project of Traditional Chinese Medicine Bureau of Jiangsu province China YB2015128Data AvailabilityReferencesThe datasets used andor analyzed during the present studyare available from the corresponding author on reasonablerequestConflicts of InterestAll authors declare that they have no conflicts of interestAuthors™ ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as cofirst authors[] M H Forouzanfar K J Foreman A M Delossantos et alœBreast and cervical cancer in countries between and a systematic analysis The Lancet vol no pp “ [] E Pelkofski J Stine N A Wages P A Gehrig K H Kimand L A Cantrell œCervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp “ [] Y Zhou W Wang R Wei œSerum bradykinin levels as adiagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2 International Journal of Oncology vol pp “ [] Y J Hu H P Zhang B Zhu H Y Chen L H Ma andY Wang œThe role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya œEfficacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp “ [] N Tanaka T Yamashita S Yamamoto œHistologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp “[] Y Watabe T Mori S Yoshimoto œCopy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp “ [] HT An S Yoo and J Ko œÎ±Actinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp “[] F Niu T Wang J Li œThe impact of genetic variants inIL1R2 on cervical cancer risk among Uygur females fromChina a casecontrol study Molecular Genetics GenomicMedicine vol no article e00516 [] W Li Y Zhao L Ren and X Wu œSerum human kallikrein represents a new marker for cervical cancer Medical Oncology vol no p [] H Shao J HC Wang M R Pollak and A Wells œÎ±Actinin4 is essential for maintaining the spreading motility andcontractility of fibroblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida œActinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp “ [] D G Thomas and D N Robinson œThe fifth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp “ screening on the diagnostic significance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp “ [] KH Wang C J Lin C J Liu œGlobal methylationsilencing of clustered protocadherin genes in cervical cancerserving as diagnostic markers comparable to HPV CancerMedicine vol no pp “ [] T Li Y Li G X Yang œDiagnostic value of combination of HPV testing and cytology as compared to isolatedcytology in screening cervical cancer a metaanalysis Journal of Cancer Research and Therapeutics vol no pp “ [] K Honda T Yamada R Endo œActinin4 a novel actinbundling protein associated with cell motility and cancer invasion The Journal of Cell Biology vol no pp “ [] E de Almeida Ribeiro N Pinotsis A Ghisleni œThestructure and regulation of human muscle αactinin Cellvol no pp “ [] D Wang X W Li X Wang œAlphaactinin4 is a possible target protein for aristolochic acid I in human kidneycellsin vitro The American Journal of Chinese Medicinevol no pp “ [] I V Ogneva N S Biryukov T A Leinsoo and I M Larina œPossible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda œThe biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim œÎ±Actinin potentiatesnuclear factor κlightchainenhancer of activated BcellNFκB activity in podocytes independent of its cytoplasmic actin binding function The Journal of BiologicalChemistry vol no pp “ [] H Shams J Golji K Garakani and M R Mofrad œDynamicRegulation of α Actinin's Calponin Homology Domains on FActin Biophysical Journal vol no pp “[] C Fang J J Li T Deng B H Li P L Geng and X TZeng œActinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp “ [] T Watanabe H Ueno Y Watabe œACTN4 copynumber increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer British Journal of Cancer vol no pp “ [] S Yamamoto H Tsuda K Honda œACTN4 gene amplification and actinin4 protein overexpression drive tumourdevelopment and histological progression in a highgrade subset of ovarian clearcell adenocarcinomas Histopathologyvol no pp “ [] M C Wang Y H Chang C C Wu œAlphaactinin is associated with cancer cell motility and is a potential biomarker in nonsmall cell lung cancer Journal of ThoracicOncology vol no pp “ [] N Okamoto H Suzuki K Kawahara œThe alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp “ 0c"
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" 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 model™s components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zˆd 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 cronbach™s 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 tool™s 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 cronbach™s 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 bartlett™s 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 truefalsedon™t know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring range˜correct™ response ˜don™t know™response ˜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 individual™s perception of his orher competence to successfully perform a behavior behavioral intention refers to a person™s perceived probability orœsubjective 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 χ2 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 bartlett™s 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 education““femalemalediplomaassociate 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 ± ˆ’ ± ± ± ± ˆ’ ± ± ˆ’ ± ˆ’ ± ± ± ˆ’ ± ± ˆ’ ± ± ˆ’ ± 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 one™sintention 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 œresearcherdesired 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 publisher™s 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 winter239“ghaffari 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 rimer“janz nk becker mh the health belief model a decade later health educ q“lp 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"
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"Gastric neoplasms containing neuroendocrine carcinoma NEC components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure NEC and mixed tumors containing NECcomponents We aimed to investigate whether there is a distinct difference in overall survival OS between gastricneoplasms containing NEC components and gastric adenocarcinomaMethods Surgically resected gastric neoplasms containing NEC components n and gastricadenocarcinomas n from January to December at Peking University Cancer Hospital wereretrospectively analysed Patients were categorized into a surgical group and a neoadjuvant group and adjustedusing pr sity score matching In the two groups gastric neoplasms containing NEC components were dividedinto pure NEC and mixed tumors with less than GHMiNEN between and GHMiNEN andmore than GHMiNEN neuroendocrine carcinoma components OS was compared between thesegroups and the gastric adenocarcinoma groupResults The OS of gastric neoplasms containing neuroendocrine NEC components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or Cox multivariable regressionanalysis suggested that tumor category neoplasms containing NEC components or gastric adenocarcinoma wasan independent risk factor for prognosis Interestingly among patients receiving neoadjuvant therapy thedifference was not significantContinued on next page Correspondence buzhaodecjcrcn jijiafuhscpkueducn Jiahui Chen Anqiang Wang and Ke Ji contributed equally to this workDepartment of Gastrointestinal Surgery Key Laboratory of Carcinogenesisand Translational Research Ministry of Education Peking University CancerHospital Institute No Fucheng Road Haidian District Beijing China 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 Cancer Page of Continued from previous pageConclusions Gastric neoplasms containing any proportion of NEC components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmsKeywords Neuroendocrine carcinoma Gastric adenocarcinoma Overall survivalBackgroundGastric neoplasms containing neuroendocrine carcinomaNEC components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure NECs and mixed tumorscontaining NEC components Every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing NEC components account for approximately “ of thesecases [ ] Given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory [“]According to the World Health anizationWHO digestive neuroendocrine tumor classificationneuroendocrine neoplasm NEN can be divided intothree categories based on Ki67 levels and mitotic counts— HPF Grade G1 Ki67 ‰ mitoses Grade G2 Ki67 ‰ ‰ mitoses‰ Grade G3Ki67 mitoses [] Meanwhile the AmericanJoint Committee on Cancer AJCC defines highly differentiated NEN as a neuroendocrine tumor NET and thepoorly differentiated NEN as a neuroendocrine carcinoma NEC based on the degree of tumor cell differentiation Generally G1 G2 and rare welldifferentiated G3NENs belong to the NETs while poorly differentiatedG3 NENs belong to NECs[ ] Gastric mixedneuroendocrinenonneuroendocrineneoplasm GMiNEN is a special type of gastric NEN that is definedas containing more than of both neuroendocrineand nonneuroendocrine components [] accountingfor approximately of all GNENs and of gastricneuroendocrine carcinomas GNECs [“] For thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition Consideringthe heterogeneity ofMiNEN and the malignancy degree of the different components in the tumor La Rosa [ ] proposeddividing MiNEN into three categories highgradeintermediategrade and lowgrade Highgrade MiNENconsists of NEC and carcinomaadenoma intermediategrade MiMEN consists of NET and carcinoma and lowgrade MiNEN consists of NET and adenoma Thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm GHMiNEN was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandGenerally the prognosis of mixed tumors is largely determined by the most malignant component Kim et al[] found that GNEC has shorter progressionfree survival PFS than gastric adenocarcinoma Huang et al[] found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components All of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis Therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone We sought tofind studies on the overall survival OS comparison between GHMiNEN and gastric adenocarcinoma butfailed Thus we think that a study of the comparison ofthe OS of GHMiNEN and gastric adenocarcinoma willprovide a valuable supplement to current research on GHMiNEN To overcome the bias caused by the differences between the covariates in the comparison we usedpr sity score matching PSM to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliableMethodsPatient selectionWe retrospectively collected patients diagnosed withgastric NENs and underwent radical resection at PekingUniversity Cancer Hospital Beijing from January to December The inclusion criteria were as follows pathologically confirmed pure NEC or tumorcontaining NEC components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup Patients diagnosedwith cM1 or cT4b before surgery or died from perioperative complications were excluded from the study 0cChen BMC Cancer Page of Patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for PSM analysesperformed The chisquared test and MannWhitney Utest were used to further verify the matching resultsFollowupWe followed the patients at least twice a year Serumtumor markers test gastroscope and computed tomography CT scans were used to reexamine patients aftersurgery Depending on the patients™ status Magneticresonance imaging MRI and Positron emission tomography computed tomography PETCT were alsoconsidered For patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationDiagnosis and classificationWe reevaluated the diagnosis and classification of GHMiNEN Mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as GHMiNEN and GHMiNENrespectively Atumor consisting of NEC is defined as pure NECAll neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the WHO classification of tumors[] Neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin Syn chromograninA CgA and neuro cell adhesion molecule CD56 orNCAM The tumor staging described in the study wasbased on the AJCC 8th Edition TNM Staging Guidelines[] All possible disagreements were discussed in ourstudy groupDefinition of variables and groupsIn this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery Patients inthe surgery group were assessed by the pTNM stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the ypTNM staging system OSrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egPr sity score matchingTo accurately compare the prognosis of GHMiNENand gastric adenocarcinoma we employed PSM to balance the differences between the two groups PSM wasperformed through the Pamatching plugin in SPSS software Logistic regression models were used toestimate pr sity scores based on gender age tumorlocation tumor size and pathological staging Given a caliper width nearest neighbor matching wasStatistical analysisAll statistical analyses were performed using SPSS statisticalsoftware IBM United States The chisquared test and MannWhitney U test were used forstatistical analysis of categorical variables and continuous variables respectively KaplanMeier method wasused for the comparison of OS The logrank test wasused to compare survival rates Multivariable Cox proportional hazards models were used to identify predictors of survival outcome P was regarded as thethreshold of significanceResultsPatient selection and PSM resultsBetween and among the patients treated atthe Gastrointestinal Cancer Center of Peking UniversityCancer Hospital a total of patients with gastric neoplasms containing NEC components met the inclusioncriteria for the study including cases of pure NECand cases of mixedtype Of these patients a total of patients received neoadjuvant therapy NEC GHMiNEN GHMiNEN GHMiNEN while the remaining patients receivedsurgery directly NEC GHMiNEN GHMiNEN GHMiNEN There were aninsufficient number of patients in group GHMiNEN group to conduct effective statistical analysisso we combined the GHMiNEN group with theNEC group for further analysis We also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery Among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly Fig Immunohistochemical specificity markers were utilizedto identify the neuroendocrine components Fig 2aSyn was expressed in almost all neoplasms containingNEC components while the positive rates ofCgA and CD56 were much lower and respectively No significant difference in the positiverate of Syn and CgA was observed between pure NEC GHMiNEN GHMiNEN and GHMiNENFig 2b c only the positive rate of CD56 was found tobe higher in the pure NEC group than that in the GHMiNEN group Fig 2dTherefore priorto OS comparison PSM wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cChen BMC Cancer Page of Fig Flow chart of patient enrolmentComparison of OS between all patients with NECcomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupBefore PSM we compared the survival curves between all patients with NEC components and patientswith gastric adenocarcinoma by the KaplanMeiermethod Fig Apparently patients with NEC components had a poorer OS than those with gastricadenocarcinoma Fig 3a p in the surgicalgroup In contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy Fig 3b p According to the proportion of NEC components patients were classifiedinto pure NEC GHMiNEN GHMiNENand GHMiNEN The OS was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison Fig 3c dFig Illustrations of immunohistochemical staining patterns in gastric neoplasms containing NEC components a An overview of the expressionof Syn CgA and CD56 in tumors containing NEC components b Syn expression in different NEC component groups c CgA expression indifferent NEC component groups d CD56 expression in different NEC component groups CD56 neuro cell adhesion molecule CgAchromogranin A NEC neuroendocrine carcinoma Syn synaptophysin Pvalue 0cChen BMC Cancer Page of Fig See legend on next page 0cChen BMC Cancer Page of See figure on previous pageFig Comparison of OS between gastric neoplasms containing NEC components and gastric adenocarcinoma a OS comparison betweengastric neoplasms containing NEC components and gastric adenocarcinoma before PSM in the surgical group b OS comparison between gastricneoplasms containing NEC components and gastric adenocarcinoma before PSM in the neoadjuvant group c OS comparison between differentNEC content groups pure NEC GHMiNEN GHMiNEN and GHMiNEN and gastric adenocarcinoma before PSM in the surgicalgroup d OS comparison between the different NEC content groups and gastric adenocarcinoma before PSM in the neoadjuvant group e OScomparison for patients in the surgical group after PSM f OS comparison for patients in the neoadjuvant group after PSM NEC neuroendocrinecarcinoma OS overall survival PSM pr sity score matchingBefore PSM significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group Table Table To balance the clinicopathological differences between the twogroups PSM was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups The detailedclinicopathological characteristics before and after PSMare shown in Table and Table As a result patients with NEC components and patients with gastric adenocarcinoma were matchedin the surgical group Table Patients with NEC components also had a poorer OS than those with gastricadenocarcinoma Fig 3e p Multivariable analysis showed that adjuvant therapy tumor category andTNM stage werefactorsTable independent prognosticTo investigate whether neoadjuvant therapy had an effect on OS patients with NEC components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group Table Interestingly KaplanMeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in OS between the two groups Fig 3f p Comparison of OS between patients with differentproportions of NEC components and patients with gastricadenocarcinomaTo investigate whether the level of NEC componentshad an effect on OS in the surgical group GHMiNEN GHMiNEN pure NEC and pure NEC plus GHMiNEN were compared with gastric adenocarcinoma after PSM The results showed that even thegroup with the lowest proportion of NEC componentsthe GHMiNEN group had a poorer OS thanadenocarcinoma Fig 4a P As expected theGHMiNEN pure NEC and pure NEC plus GHMiNEN groups each with relatively high proportionsof NEC components had worse OS than the gastricadenocarcinoma group Fig 4bd P Detailed clinical information after matching isshown in Additional file Tables S1S4PSM was also performed in the neoadjuvant group Incontrast to the results of the surgery group in the pureNEC group containing the highest proportion ofNEC componentstill no significantdifference in OS from gastric adenocarcinoma Fig5d The other three groups with lower NEC contentwere also notfrom gastricadenocarcinoma in terms of OS Fig 5ac Detailedclinicopathologicaland afterPSM are shown in Additional file Tables S5S8characteristics beforethere wassignificantly differentDiscussionAmong gastric neuroendocrine neoplasms the tumorcontaining NEC components is a special type includingpure NEC and mixed tumor containing NEC components The incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated GNENs [ ]received neoadjuvantIn previous study Kim found that in patientschemotherapywho had notprogressionfree survivalPFS of pure GNEC waspoorer than that of gastric adenocarcinoma while thePFS of mixedtype tumors was not significantly differentIn Kim™sfrom that of gastric adenocarcinoma []study the mixed type was defined as NET mixed withgastric cancer rather than NEC NET is much less malignant than NEC [ ] This may be the reason whythere was no significant difference in OS between mixedtype and gastric adenocarcinomas In addition mixed tumors with less than or more than of NEC components were not included in that study which webelieve was a deficit of the study PFS is an important indicator for evaluating prognosis in many cases it can reflect the trend of OS Based on Kim™s research resultswe regarded tumors containing NEC components as awhole and found that the OS of these tumors was poorerthan that of adenocarcinoma in the surgical group Inthe comparison of OS between mixed tumors with different proportions of NEC components and gastricadenocarcinoma the results for pure NEC cases wassimilar to Kim™s While the OS of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in Kim™s study Cox multivariable regression analysis showed thattumor categoryneoplasm with NEC component or adenocarcinoma 0cChen BMC Cancer Page of Table Comparison of clinicopathological characteristics before and after PSM in surgical groupPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n P valueMatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm‰¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomy ± ± Proximal gastrectomy Surgical procedure LaparoscopicT stageT1T2T3T4N stageN0N1N2N3M stageM0M1pTNM stageIIIIIIIV Gastricadenocarcinoman ± ± ± ± P value Gastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Table Comparison of clinicopathological characteristics before and after PSM in neoadjuvant groupMatched comparisonPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm‰¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedure LaparoscopicT stageT0T1T2T3T4N stageN0N1N2N3M stageM0M1ypTNM stageIIIIIIIV ± ± Gastricadenocarcinoman ± ± P valuePatients with NECcomponents n ± ± Page of P valueGastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Page of Table Univariate and multivariate analyses of survival after PSM in surgical groupPatient CharacteristicsUnivariate analysisHR CI“Multivariate analysisHR CIP valueAge yearGendermale vs femaleBMIAdjuvant therapyYes vs NoTumor size‰¥ cm vs cmTumor categoryCarcinoma with NEC component vsGastric adenocarcinoma vsType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedureLaparoscopic vs TNM stageIIIIIIIVP value“““ ““““““““““ “ ““““““““tumor size and TNM staging were independent risk factors for prognosis This suggests that the prognosis ofgastric neoplasms with NEC components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of NEC components can alsoimpair prognosis which challenges the current cutoffvalue of The proportion of each component that must theoretically be greater than was set in [] Andsince WHO has also adopted this standard to define MiNEN [] This largely avoids the overdiagnosisof MiNEN in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges In additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines [] Nevertheless it is now being questionedby an increasing number of scholars The componentsin mixed tumors are not evenly distributed For large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely []Park compared the OS between tumors with morethan NEC components and gastric adenocarcinomawith or without less than NEC and they found thattumors with an NEC composition of more than hada worse prognosis This suggests that even a small proportion of malignant components can affect prognosis[] While in Park™s study for unknown reasons the authors did not compare the prognosis of mixed tumorswith NEC components less than with gastricadenocarcinomas directly nor did they compare allNECcontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyIn our study we regarded tumors containing NECcomponents as a whole and found that the OS of thesetumors was poorer than that of adenocarcinoma in thesurgical group In addition we also found that the OS ofmixed tumors with less than between and more than NEC components or pure NEC wasworse than that of gastric adenocarcinoma Analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of Syn and CgAbetween different NEC content groups only the positiverate of CD56 was found to be higher in the pure NECgroup than that in the GHMiNEN group Therole of CD56 in the diagnosis of NEC is still controversial However Syn and CgA are two wellrecognized 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC and gastric adenocarcinoma in the surgical group aOverall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparison between GHMiNEN andgastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastric adenocarcinoma d Overall survivalcomparison between pure NEC alone and gastric adenocarcinomamarkers Therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing NEC componentsStudies on the molecular mechanism of pathogenesisshow that NEC components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity LOH and mutations at multiple loci and key oncogenes such as TP53 APC and RBgenes All these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[“] Moreoverin the WHO definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid [] no minimumpercentage for either ingredient is established Thereforewe believe that mixed tumors containing NEC components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma We propose considering mixed tumorscontaining NEC components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiesPreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] In a retrospective study involving patientsMa et alfound that neoadjuvant chemotherapy improves the survival of patients with NEC and HMiNENof the stomach [] Van der Veen reported that 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC components and gastric adenocarcinoma in theneoadjuvant group a Overall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparisonbetween GHMiNEN and gastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastricadenocarcinoma d Overall survival comparison between pure NEC and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing NEC components [] However because only eight patients wereincluded in the neoadjuvant group Van™s results arequestionable In our study among patients receivingneoadjuvanttherapy no significant difference in OSbetween mixed tumor and gastric adenocarcinoma wasobserved Even for the pure NEC group with the highestNEC contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsAlthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research In addition we proposed treatingall gastric neoplasms containing NEC components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms In the futurewe will conduct more genomics studies to confirmour ideas This study also has its limitations Due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens As a retrospective study despite our performing PSM in advance selection biascannot be completely avoided In addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cChen BMC Cancer Page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with NEC componentless than so we could only treat all mixed tumors withNEC component as a wholeConclusionsOur study demonstrated that gastric neoplasms withNEC components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies Therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients Neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12885020072817Additional file Table S1 Comparison of clinicopathologicalcharacteristics before and after PSM of 30GHMiNEN patients insurgical group Table S2 Comparison of clinicopathologicalcharacteristics before and after PSM of GHMiNEN patients in surgicalgroup Table S3 Comparison of clinicopathological characteristics beforeand after PSM of 70GHMiNEN plus pure NEC patients in surgicalgroup Table S4 Comparison of clinicopathological characteristics beforeand after PSM of pure NEC patients in surgical group Table S5 Comparison of clinicopathological characteristics before and after PSM of 30GHMiNEN patients in neoadjuvant group Table S6 Comparison ofclinicopathological characteristics before and after PSM of GHMiNEN patients in neoadjuvant group Table S7 Comparison of clinicopathologicalcharacteristics before and after PSM of 70GHMiNEN plus pure NECpatients in neoadjuvant group Table S8 Comparison of clinicopathological characteristics before and after PSM of pure NEC patients in neoadjuvant groupAbbreviationsAJCC American Joint Committee on cancer CT Computed tomography GHMiNEN Gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm GNEC Gastric neuroendocrine carcinoma HPF High power fieldMiNEN Mixed neuroendocrinenonneuroendocrine neoplasmNEC Neuroendocrine carcinoma NEN Neuroendocrine neoplasmNET Neuroendocrine tumor MRI Magnetic resonance imaging OS Overallsurvival PETCT Positron emission tomography computed tomographyPFS Progressionfree survival PSM Pr sity score matching WHO WorldHealth anizationAcknowledgmentsThanks to Dr Zhongwu Li of the Department of Pathology Peking UniversityCancer Hospital and his colleagues for their assistance in pathologicaldiagnosis and review Thanks to all colleagues in the Department ofGastrointestinal Surgery of Peking University Cancer Hospital and Dr JiangHong from the Statistics Department for their assistance in this studyAuthors™ contributionsAll authors contributed to the study conception and design JC performeddata collection and wrote the manuscript AW wrote and t revised hemanuscript KJ helped with statistical analysis and prepared the illustrationsZB edited the manuscript JJ conceived the study and reviewed themanuscript All authors read and approved the final manuscriptFundingThis work was supported by the National Science Foundation for YoungScientists of China Beijing Youth Talent Plan QML20191101 andScience Foundation of Peking University Cancer Hospital “ Thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the Ethics Committee of Peking UniversityCancer Hospital and the patients™ written consent was also obtained Writteninformed consent for publication was obtained and stored in PekingUniversity Cancer HospitalConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived May Accepted August ReferencesBray 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 “ Matsubayashi H Takagaki S Otsubo T Iiri T Kobayashi Y Yokota T et alAdvanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy Gastric Cancer “Park JY Ryu MH Park YS Park HJ Ryoo BY Kim MG Prognosticsignificance of neuroendocrine components in gastric carcinomas Eur JCancer “La Rosa S Inzani F Vanoli A Klersy C Dainese L Rindi G Histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms Hum Pathol “Ishida M Sekine S Fukagawa T Ohashi M Morita S Taniguchi H et alNeuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis Am J Surg Pathol“Rayhan N Sano T Qian ZR Obari AK Hirokawa M Histological andimmunohistochemical study of composite neuroendocrineexocrinecarc
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"unrestricted use distribution and reproduction in any medium provided the original work is properly citedPurpose The present study was aimed at determining the serum levels of actinin4 ACTN4 in cervical cancer CC andinvestigating the diagnostic and prognostic value of serum ACTN4 in CC Materials and Methods We included CC patients cervical intraepithelial neoplasia CIN patients and healthy women Serum ACTN4 levels were assessed using an ELISAmethod A receiver operating characteristic ROC curve was performed to evaluate the diagnostic value of serum ACTN4 Thesurvival curves were used to display the overall survival distributions Results Serum ACTN4 levels in CC patients were ± pgmL which is significantly higher than those in CIN patients ± pgmL P and those in healthycontrols ± pgmL P The ROC analysis demonstrated that the area under the curve AUC of ACTN4 was 95CI “ with sensitivity of and specificity of Serum ACTN4 levels were associated with theFIGO stage lymph node metastasis and lymphovascular space invasion of CC all P The survival curve suggested thathigh serum ACTN4 levels were related to poor prognosis Conclusion Our findings suggest that serum ACTN4 levels may bevaluable diagnostic and prognostic biomarkers for CC IntroductionCervical cancer CC is the second most common femalemalignancy globally and it is the most common femalemalignancy in developing countries which has high morbidity and mortality rates [] In recent years the incidence ofCC has increased greatly in young women under the age of [] Despite great advances in surgical and adjuvant therapy the overall survival of CC patients especially that ofadvanced patients is still very poor [] At present a Papsmear combined with an HPV test has been used for the earlyscreening of cervicalthe screeningmethods are invasive and costly leading to lower screeningcoverage in China [] Previous studies have reported thatthe human papillomavirus HPV screening results have arelatively high falsepositive rate and a relatively low specificity [ ] In addition the results of TCT interpretation byfilmreading doctors are uneven which might cause somelesions Howevermisleadingness in the choices of prevention measures andtreatment for CC [] Noteworthily when applying the sametreatment plan to patients with similar pathological types theefficacy and prognosis are quite diï¬erent Therefore it is necessary to identify new biomarkers directly related to the progression and prognosis of CCAlphaactinins ACTNs are actinbinding proteins inthe spectrin gene superfamily [] which are known to becrosslinked with filamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1“which are present in humans and other mammals [“]ACTN4 is encoded by the ACTN4 gene and is widelyexpressed in many tissues especially in glomerular podocytes[] ACTN4 has an actinbinding domain at the Nterminus and ACTN4 monomers can form a homodimer throughreverse binding forming a dumbbellshaped structure []As an actinbinding protein ACTN4 is closely related to 0cDisease Markersenhancing cell viability and tumor invasion and metastasis[] Recent researches have reported that the expression ofACTN4 is significantly elevated in multiple cancers including breast cancer [] pancreatic cancer [] ovarian cancer[] and lung cancer [] In addition the ACTN4 levels aremarkedly associated with the poor prognosis of lung cancer[ ] thyroid cancer [] and salivary gland carcinoma[] An [] have found that the expression level ofACTN4 in human cervical tumors is dramatically higherthan that in normal cervical tissues Their finding demonstratedepithelialtomesenchymal transition and tumorigenesis by regulatingSnail expression and the Akt pathway in CC [] Thereforethe expression of ACTN4 in cervical tissues may be used inthe clinical diagnosis and prognosis prediction of CCthat ACTN4promotestheHowever up to now the significance of the serumACTN4 levels in CC has not been evaluated Hence in thecurrent study the serum levels of ACTN4 in patients withCC were measured In addition we estimated the potentialdiagnostic and prognostic value of serum ACTN4 expressionin CC Materials and Methods Study Population A retrospective study was designed toevaluate serum actinin4 as a biomarker for CC Between July and June newly diagnosed female CC patientsand newly diagnosed female cervical intraepithelial neoplasia CIN patients who received treatment at Huai™anMaternal and Child Health Care Hospital Huai™an JiangsuChina were recruited The diagnoses of all patients were verified by the histopathological examination The patients withother types of tumor or autoimmune atherosclerotic andhematologic diseases were excluded The mean age of CCpatients was years with a range of years Meanwhile healthy women with no evidence of neoplasmsand other serious diseases were enrolled from the physicalexamination center in the same hospital There was no significant diï¬erence in age among the CC CIN and healthy control groups This study was consistent with the Helsinkideclaration and was authorized by the Ethics Committee ofHuai™an Maternal and Child Health Care Hospital approvalnumber H20130504 All participantssigned writteninformed consent Clinicopathologic Feature Collection and FollowUp Byreviewing the medical records we collected the clinicopathologic characteristics of the patients including age at diagnosis pathological type FIGO stage tumor diï¬erentiationpelvic lymph node metastasis tumor size and lymphovascular space invasion The CC patients were classified based onthe revised FIGO staging system for CC in The tumorsize was the maximum tumor diameter determined by agynecologic oncologist during pelvic examination Thepatients in stage 1A1 received hysterectomy the patients instages IB1 and IIB received radical hysterectomy and pelviclymph node dissection the patients with ‰¥stage IIB receivedradiotherapy or radiotherapy combined with chemotherapyA regular telephone followup was conducted after treatmentto obtain the overall survival OS time of CC patients andthe OS was defined as the time from diagnosis to death orthe last followup The followup was in accordance withthe FIGO guidelines Blood Sample Collection and Detection of Serum Actinin and SCCA A mL peripheral blood sample from eachpatient was collected before receiving any treatment Afterstanding at room temperature for minutes the blood samples were centrifugated at gmin for min and then°the supernatant was stored at ˆ’C until further usageThe serum actinin4 concentration was measured by a quantitativeELISAmethod Uscn Life Science Inc Wuhan China The levelsof SCCA in serum were determined using an ELISA kitRD Systems Minneapolis MN The detection of all samples was strictly in accordance with the instructions providedby the manufacturer and was performed in duplicatesenzymelinked immunosorbentassay Statistical Analysis All statistical analyses were conducted by using SPSS and GraphPad Prism The continuous data following normal distribution were expressed asthe mean ± standard deviation°SDž A ttest was used tocompare serum ACTN4 levels between the two subgroupsof each clinicopathological parameters and the serumACTN4 levels of CC patients CIN patients and healthy controls were compared by the SNKq test Receiver operatingcharacteristic ROC curves were performed to assess thediagnostic value of serum ACTN4 levels for diï¬erentiatingCC patients from CIN patients and healthy controls TheKaplanMeier method and logrank test were used to plotsurvival curves The Cox proportional hazards models in univariate and multivariate analyses were used for evaluating theprognostic value of serum ACTN4 expression A twotailed Pvalue was considered to be statistically significant Results Serum ACTN4 Levels Are Higher in Patients with CCSerum concentrations of ACTN4 were detected to rangefrom to pgmL with a mean ±SD of ± pgmL for CC patientsto range from to ngmL with a mean ±SD of ± pgmL forCIN patients and to range from to ngmL witha mean ±SD of ± pgmL for healthy controlsSerum ACTN4 levels in CC patients were significantly higherthan those in CIN patients and healthy controls P However no significant diï¬erence in serum ACTN4 wasfound between CIN patientscontrolsP as shown in Figure and healthy The Diagnostic Value of Serum ACTN4 Levels for CC Wenext used ROC curve analysis to estimate the diagnostic valueof serum ACTN4 expression for CC The ROC curve showedthat the serum levels of ACTN4 were robust for discriminating CC patients from benign and healthy control subjectswith an area under the curve AUC value of 95CI “ as demonstrated in Figure index we usedAccordingto maximum Youden™s 0cDisease MarkersŽŽlymph node metastasis were the independent prognostic factors for CC all P Table Lmgp NTCAnsCCCINCON DiscussionCervical cancer is a heterogeneous disease with complicatedetiology Genetic and environmental factors play a crucialrole in the pathogenesis of CC [] Although the diagnosisand prognosis of CC have improved greatly over the pastfew decades it is necessary to improve early detection andscreening methods to determine additional promising circulating biomarkers for better patient selection and more personalized treatments [] As far as we know this studyrepresented the first eï¬ort to evaluate the serum expressionof ACTN4 as a new biomarker for CCAs an actinbinding protein ACTN4 can participate inregulating cell migration invasion and metastasis via regulating the actin filament flexibility at the leading edge ofinvading cancer cells [ ] ACTN4overexpressing cancercells have the potential to metastasize because the overexpression of ACTN4 protein in cancer cells can stimulate thedynamic reconstruction of the actin cytoskeleton [] Upto now numerous studies have reported the associationbetween ACTN4 and multiple cancers Okamoto []observed that ACTN4 is expressed in smallcell lung cancerNSCLC and it had a significant correlation with invasionand distant metastasis Additionally ACTN4 was reportedto be a potential predictive biomarker for the efficacy of adjuvant chemotherapy in patients with NSCLC [] Watabe [] revealed that the copy number increase of ACTN4is a novel indicator for poor overall survival of patients withsalivary gland carcinoma and the copy number variationwould aï¬ect the expression of protein A recent study demonstrated that serum ACTN4 levels were dramatically elevated in patients with breast cancer when compared tohealthy controls and serum ACTN4 may be an eï¬ective clinical indicator for diagnosing or predicting the clinical outcomes of breast cancer patients [] In addition ACTN4was proven to be associated with the pathogenesis of CCAn [] proposed a novel mechanism for epithelialtomesenchymal transition and tumorigenesis in CC whichcould be induced by ACTN4 through regulating Snail expression and βcatenin stabilization Hence it is significant toinvestigate the role of serum ACTN4 in CCIn the current study we observed that serum levels ofACTN4 in CC patients were statistically higher than thosein CIN patients and those in healthy controls Howeverserum ACTN4 levels were not significantly diï¬erent betweenthe CIN group and the control group It was shown thatserum ACTN4 expression could strongly diï¬erentiate CCpatients from CIN patients and healthy controls The ROCanalysis demonstrated that the AUC of ACTN4 was and at the optimal cutoï¬ of pgmL the sensitivity andspecificity were respectively and suggestingthat serum ACTN4 might be a potential diagnostic biomarker for CC In a recent study which included Chinesewomen Hu [] reported that the sensitivity and specificity of HPV screening in the diagnosis of CC were and The sensitivity of the HPV test was a litter higherFigure The serum ACTN4 levels in CC patients CIN patientsand healthy controls ˆ—P pgmL as the cutoï¬ value and the sensitivity and specificity were and respectively Association between Serum ACTN4 Levels andClinicopathological Parameters of CC Patients We furtherinvestigated the correlations between serum levels of ACTN4and clinical pathological data of CC patients and theresults are demonstrated in Table We observed that serumACTN4 levels were related to the FIGO stage lymph nodemetastasis and lymphovascular space invasion all P Nevertheless no significant association was found betweenserum ACTN4 levels and age pathological type diï¬erentiation degree and tumor size in CC patients all P Survival Analysis of Serum ACTN4 Levels in CC Duringthe followup period nine CC patients were lost and thefollowed up rate is Finally the prognostic value ofserum ACTN4 was assessed in patients The patients werefollowed up to December The range of followup timewas to months with the median time of months andmean time of months According to the median serumlevels of ACTN4 in CC patients pgmL the CCpatients were divided into the high ACTN4 level group pgmL N and low ACTN4 level group‰¥ pgmL N The estimated 5year OS of patientswith high serum ACTN4 levels and low serum ACTN4 levelswere and respectively The KaplanMeier survival curve and logrank test indicated that CC patients withhigh serum ACTN4 levels had a worse prognosis than thosewith low serum ACTN4 levels P Figure Univariate Cox regression analyses showed that theserum ACTN4 levels P FIGO stage P diï¬erentiation degree P lymph node metastasisP and lymphovascular space invasion P had significant prognostic value for OS Multivariate analysiswas further performed to evaluate the prognostic value ofserum ACTN4 as an independent factor for CC All the statistically significant factors from univariate analyses wereincluded and the results indicated that the FIGO stage and 0cDisease MarkersytivitisneS ˆ’ specificityFigure ROC curve analysis assessed the diagnostic performance of serum ACTN4 in CC The AUC was P Table Serum ACTN4 levels in CC patients according toclinicopathological parametersParametersAge years‰¤Pathological typeSquamous cell carcinomaAdenocarcinomaFIGO stageIA1IB1‰¥IB2Diï¬erentiationWell and moderatelydiï¬erentiatedPoorly diï¬erentiatedLymph node involvementNegativePositiveTumor size‰¤Lymphovascular space invasionNegativePositiveN ACTN4pgmLP ± ± ± ± ± ± ± ± ± ± ± ± ± ± than that of serum ACTN4 detection though the specificityof serum ACTN4 detection was well above that of the HPVtest Hence comparing with the HPV test in diagnosingCC detecting serum ACTN4 has some advantages Furthermore serum ACTN4 levels have been indicated to be a greatbiomarker for diagnosing multiple cancers Fang [] intheir study reported that serum ACTN4 was a promisingindicator for diagnosing breast cancer with the AUC of Wang [] used ACTN4 expression in peripheralblood to diï¬erentiate NSCLC patients from healthy individuals in two groups of participants and they obtained bothsatisfactory eï¬ects Furthermore we investigated the correlation between serum ACTN4 and clinical characteristics ofCC patients The serum ACTN4 levels were significantlyassociated with the FIGO stage lymph node metastasis andlymphovascular space invasion of CC which suggests thatACTN4 could contribute to the development invasion andmetastasis of CC In addition our results indicated that highACTN4 levels were associated with the poor survival of CCpatients In the multivariate analysis although ACTN4 levelsdid not reach the statistical significance it still seems to beable to influence the OSHowever several limitations in the present study should betaken into consideration First the sample size was relativelysmall which was likely to reduce the statistical power of ourresults Second we only explored the relationship betweenserum ACTN4 and OS and other prognostic indicators werenot examined due to the incomplete data which needs to beimproved in the future Third this study was a primary studyto determine the clinical significance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the specific molecular mechanisms remain unclear Hence further experimentsshould be conducted to elucidate the mechanismsIn conclusion our study showed that serum ACTN4levels were increased in CC patients and were related to the 0cDisease Markers lavivrus muCLog rank P Overall survival monthsLow ACTN4 groupHigh ACTN4 groupFigure KaplanMeier curve compared OS of CC patients with high serum ACTN4 levels versus those with low serum ACTN4 levelsTable Univariate and multivariate Cox regression analysis of OS in CC patientsUnivariate CIVariablesAge vs ‰¤ yearsPathological type squamous cell carcinoma vs adenocarcinomaFIGO stage ‰¥IB2 vs IA1IB1Diï¬erentiation poorly diï¬erentiated vs well and moderately diï¬erentiated “Lymph node involvement positive vs negativeTumor size vs ‰¤ cmLymphovascular space invasion positive vs negativeSerum ACTN4 levels high vs low levelsHR “ “ “ “ “ “ “ “ “ “ “ “P”””Multivariate CIPHRFIGO stage lymph node metastasis and lymphovascularspace invasion of CC patients In addition serum levels ofACTN4 have great diagnostic and prognostic value in CCNevertheless further studies with a larger sample size shouldbe carried out to confirm our resultsAcknowledgmentsWe thank all the patients and blood donors who participatedin our study This study was funded by grants from the Science and Technology Project of Traditional Chinese Medicine Bureau of Jiangsu province China YB2015128Data AvailabilityReferencesThe datasets used andor analyzed during the present studyare available from the corresponding author on reasonablerequestConflicts of InterestAll authors declare that they have no conflicts of interestAuthors™ ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as cofirst authors[] M H Forouzanfar K J Foreman A M Delossantos et alœBreast and cervical cancer in countries between and a systematic analysis The Lancet vol no pp “ [] E Pelkofski J Stine N A Wages P A Gehrig K H Kimand L A Cantrell œCervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp “ [] Y Zhou W Wang R Wei œSerum bradykinin levels as adiagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2 International Journal of Oncology vol pp “ [] Y J Hu H P Zhang B Zhu H Y Chen L H Ma andY Wang œThe role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya œEfficacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp “ [] N Tanaka T Yamashita S Yamamoto œHistologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp “[] Y Watabe T Mori S Yoshimoto œCopy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp “ [] HT An S Yoo and J Ko œÎ±Actinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp “[] F Niu T Wang J Li œThe impact of genetic variants inIL1R2 on cervical cancer risk among Uygur females fromChina a casecontrol study Molecular Genetics GenomicMedicine vol no article e00516 [] W Li Y Zhao L Ren and X Wu œSerum human kallikrein represents a new marker for cervical cancer Medical Oncology vol no p [] H Shao J HC Wang M R Pollak and A Wells œÎ±Actinin4 is essential for maintaining the spreading motility andcontractility of fibroblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida œActinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp “ [] D G Thomas and D N Robinson œThe fifth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp “ screening on the diagnostic significance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp “ [] KH Wang C J Lin C J Liu œGlobal methylationsilencing of clustered protocadherin genes in cervical cancerserving as diagnostic markers comparable to HPV CancerMedicine vol no pp “ [] T Li Y Li G X Yang œDiagnostic value of combination of HPV testing and cytology as compared to isolatedcytology in screening cervical cancer a metaanalysis Journal of Cancer Research and Therapeutics vol no pp “ [] K Honda T Yamada R Endo œActinin4 a novel actinbundling protein associated with cell motility and cancer invasion The Journal of Cell Biology vol no pp “ [] E de Almeida Ribeiro N Pinotsis A Ghisleni œThestructure and regulation of human muscle αactinin Cellvol no pp “ [] D Wang X W Li X Wang œAlphaactinin4 is a possible target protein for aristolochic acid I in human kidneycellsin vitro The American Journal of Chinese Medicinevol no pp “ [] I V Ogneva N S Biryukov T A Leinsoo and I M Larina œPossible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda œThe biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim œÎ±Actinin potentiatesnuclear factor κlightchainenhancer of activated BcellNFκB activity in podocytes independent of its cytoplasmic actin binding function The Journal of BiologicalChemistry vol no pp “ [] H Shams J Golji K Garakani and M R Mofrad œDynamicRegulation of α Actinin's Calponin Homology Domains on FActin Biophysical Journal vol no pp “[] C Fang J J Li T Deng B H Li P L Geng and X TZeng œActinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp “ [] T Watanabe H Ueno Y Watabe œACTN4 copynumber increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer British Journal of Cancer vol no pp “ [] S Yamamoto H Tsuda K Honda œACTN4 gene amplification and actinin4 protein overexpression drive tumourdevelopment and histological progression in a highgrade subset of ovarian clearcell adenocarcinomas Histopathologyvol no pp “ [] M C Wang Y H Chang C C Wu œAlphaactinin is associated with cancer cell motility and is a potential biomarker in nonsmall cell lung cancer Journal of ThoracicOncology vol no pp “ [] N Okamoto H Suzuki K Kawahara œThe alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp “ 0c"
2
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0“ in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global moran™s index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of pm25 and pm10 in “ respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range “ “ “ pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0“ was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0“ was associated with a increase of daily confirmed new cases wuhan rr ci1032“ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p χ2 p a μgm3 increase in pm25 was associated with a “ increase in fatality rate χ2 p a μgm3 increase in pm10 was associated with a “ increase in fatality rate no2 a 10μgm3 no2 increase lag0“ was associated with a increase in daily confirmed new cases wuhan rr ci “ xiaogan rr ci “ huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci “ estimate on deaths value cid0 ci “ kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearman™s one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci “ and deaths cid0 ci “ low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years “ and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0“ in no2 is associated with a ci “ increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053“ xiaogan rr ci “ but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations “ pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol “ httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res “ httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health “ httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med “ httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health “ https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res “ https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 “ conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest 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 environmentalresearch19120201101297 0cc cop
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" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since “ beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on people™s health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of today™sdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical 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 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and [“]in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rrˆ’¾ ¾ pexp 02 rrˆ’°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso “ times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm [“]the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in “ sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in “disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungary™s gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the country™s gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of “ billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that “ of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary “ in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary “medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ “] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amountˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ ˆ’ˆ’ˆ’ ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ total amountˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ˆ’ˆ’ˆ’ˆ’inactivityˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ ˆ’ ˆ’ ˆ’ˆ’ˆ’ ˆ’ ˆ’ 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the population™s health status is responsible for about “ ofeconomic growth [“]in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases™ burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifa™s depression costs account foronly “ in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto people™s daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects “ such as the high costof sports injuries high rates of childhood illness “ haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since “ beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifa™s colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors™ contributionspa was the leader of the complete research coordinated the different coauthors™ work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev “reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med “ who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379“tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab “rishiraj n inactivity a bad œhabit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle “ gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev “ acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed “ hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©g“befektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health “katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj “ 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed “ andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c
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"In our newly developed approach for the fast generation of tumor cohorts we have overcome this obstacle as exemplified for three GEMMs; two lung cancer models and one mesothelioma model. Three elements are central for this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation. As proof-of-principle we demonstrate that MycL1 is a key driver gene in Small Cell Lung Cancer. cancer chimeras embryonic stem cells mouse models MycL1 Introduction The toolbox for generating genetically engineered mouse models (GEMMs) has been steadily growing over the last couple of years. Most of the new technologies deal with genetic modification methods of embryonic stem cells (ESCs) for instance using a recombinase or an integrase to introduce genetic elements in predefined loci (Belteki et al 2003; Beard et al 2006; Seibler et al 2007) or Zinc finger TAL effector and RNA-guided nucleases to create mutant alleles with high flexibility and ease (Urnov et al 2010; Miller et al 2011; Cong et al 2013). Although these technologies increase versatility they provide minor time gains as the time spent to genetically engineer ESCs is often dwarfed by the time required to cross the resulting mice to existing GEMMs in order to obtain the final experimental cohort. This is particularly an issue in cancer research as spontaneous tumor development in GEMMs often requires the (in)activation of multiple genetically modified oncogenes and tumor suppressor alleles. For instance we have developed GEMMs for Small Cell Lung Cancer (SCLC) and mesothelioma that have four and six conditional alleles respectively (Meuwissen et al 2003; Jongsma et al 2008). This genetic complexity has hampered the use of GEMMs to study additional candidate cancer genes for their role in tumor initiation and progression. This problem will only become more pressing now genome-wide association studies and genetic screens result in the identification of an increasing number of candidate genes whose roles in tumorigenesis need confirmation in relevant in vivo models (Chin et al 2011). One strategy to accelerate target gene validation in mouse models is to apply the CRISPR/Cas system in zygotes for the one-step generation of animals carrying mutations in multiple genes (Wang et al 2013). Though extremely powerful this technique needs to be further developed to allow for the controlled introduction of transgenes and/or conditional alleles. Also off-target effects are likely to occur and need to be taken into account (Fu et al 2013). We and others have presented an alternative and well-controlled strategy by re-deriving ESCs from well-established and validated GEMMs and use these GEMM-ESCs as the basis for further genetic engineering either by classic gene targeting gene editing or recombinase-mediated transgene integration (Nichols et al 2009; Huijbers et al 2011; Premsrirut et al 2011). These GEMM-ESCs contain the same genetic modifications as present in the original model plus the newly introduced genetic modification for instance a frequently observed point mutation in a tumor suppressor gene or a transgene with conditional expression of an oncogene that is often amplified or otherwise overexpressed in a particular tumor type. These modified GEMM-ESCs can be used to generate high quality chimeras that are likely equally susceptible to tumor induction as the original GEMM and serve as a defined experimental cohort differing only by the introduced modification. Main advantages of this approach are speed and flexibility as it permits comparative analysis of phenotypic consequences of different genes and allelic series in a particular GEMM. Instead of crossing the chimeric mice to the desired strain and genetic background ready-to-use GEMMs can now be produced on-demand. This reduces both costs and total number of mice needed per experiment as establishing and maintaining a large breeding colony is expensive and always leads to surplus animals that cannot be used in experiments. Furthermore genetic drift is prevented as the same GEMM-ESCs lie at the basis of each experimental cohort for a particular cancer type. This approach also allows for the establishment of a GEMM-ESC bank for distribution ESCs with complex genotypes; a resource that likely gives a new impulse to the generation of custom-made mouse models either for preclinical use or cancer gene validation. The feasibility of the GEMM-ESC production approach depends on reliable procedures and robust quality controls including (i) ESC culture procedures that guarantee maintenance of pluripotency; (ii) monitoring of the genomic stability of ESCs; (iii) procedures for routine production of chimeras with a major contribution of the GEMM-ESCs to different tissues. The chimeric lines should also be germline-competent to facilitate the production of permanent lines if desirable. Here we present the performance of the GEMM-ESC approach based on three different GEMMs two models for lung cancer and one for mesothelioma. We employed the Flp-mediated integration technology (Flp-in) as proof-of-concept for genetic engineering of GEMM-ESCs. Finally we apply the GEMM-ESC approach to validate Mycl1 as a bona fide oncogene in SCLC. Results ESC culture and pluripotency The first step in the GEMM-ESC approach is the derivation of ESC from the desired GEMMs which are often backcrossed to a specific strain background such as C57BL/6J (black coat color) or FVB/n (white coat color). Using classical culture conditions ESC derivation can be achieved for permissive strains such as 129 and C57BL/6N but various strains are thought to be non-permissive (Kawase et al 1994; Schoonjans et al 2003). New culture protocols now permit the derivation of ESCs from refractory strains (Ying et al 2008). In these protocols culture media containing fetal bovine serum (FBS) and feeder cells are replaced by defined N2B27 medium supplemented with two inhibitors (2i): the MEK1 inhibitor PD0325901 which effectively blocks MEK/ERK signaling thereby preventing ESC differentiation and the GSK3 inhibitor CHIR99021 which acts as a Wnt agonist thereby stimulating growth of ESCs. We used this 2i medium to derive ESCs from wild-type C57BL/6J and FVB/n strains and obtained 4 and 12 ESC clones respectively (). Culturing of ESCs in 2i medium improved their overall quality. Expression analysis of the core ESC transcription factors Nanog Oct4 (also known as Pou5f1) and Sox2 in wild-type 129/Ola ESCs showed a higher percentage of na¯ve Nanogpos;Oct4pos;Sox2pos ESCs under the new culture conditions as compared to the classic conditions (supplementary Fig S1). These results demonstrate that 2i medium is more effective in maintaining ESCs in a na¯ve undifferentiated state. ESC derivation. Genotype Strain Embryos ICM ESC clones Efficiencya(%) Male Female Wt C57LB/6J 30 17 4 13.3 4 0 Wt FVB/n 35 22 12 34.3 6 6 Kras LSL-G12D C57BL/6J 57 24 8 14.0 8 0 Rb1F/F ;Trp53F/F FVB/n;129/Ola 145 63 13 8.9 13 0 Nf2F/F ;Trp53F/F ;Cdkn2a*/* FVB/n;129/Ola 65 31 5 7.7 5 0 Rb1F/F ;Trp53F/F ;Col1A1-frt FVB/n;129/Ola 32 26 5 15.6 3 2 a ESC derivation efficiency: percentage of isolated embryos resulting in established ESC clones. Quality controls of derived ESCs The quality of derived ESCs was assessed on the basis of three criteria: expression of stem cell markers chimeric contribution and germline transmission. The first criterion was determined by FACS profiling. ESC clones from either C57BL/6J (clone 1.4) or FVB/n (clone 1.3) showed robust expression of Nanog Oct4 and Sox2 in the majority of cells (Fig 1A and B). To test chimeric contribution of the derived ESCs each clone was injected into host blastocysts or morulae to generate chimeric animals that were scored for their coat-color chimerism. When ˜black™ C57BL/6J ESCs were injected in ˜white™ FVB/n hosts the coat color of the resulting chimeras was scored for the absence of white fur and vice versa. We compared three injection strategies for the wild-type C57BL/6J ESC clone: (i) blastocysts injected with 12“15 cells with direct implantation into foster mothers; (ii) morulae injected with 4“8 cells with direct implantation; (iii) morulae injected with 4“8 cells with implantation after overnight culture of the embryos in embryo culture medium (Fig 1C). Based on the coat color contribution the latter strategy clearly outperformed the other two with 100% coat color contribution for several mice. Similar results were obtained for a wild-type FVB/n ESC clone (Fig 1D). The increase in coat color contribution came at a price as the number of liveborn chimeras relative to the total number of implanted embryos was lower for the morula injections than for the blastocyst injections (Fig 1E and F and supplementary Table S1). This drop in viability is likely the result of aberrant embryonic development leading to resorption in utero or in milder cases the birth of runted animals. In addition some foster mice implanted with morulae injected with FVB/n or FVB/n;129/Ola ESCs were unable to give natural birth and caesarean section was required. To ensure a practical workflow we decided to inject C57BL/6J ESC clones into morulae (FVB/n) as for this background the benefit of improved chimerism outweighed the complications. In case of FVB/n or mixed FVB/n;129/Ola ESC clones we used more fail-safe blastocyst injections (C57BL/6N). Figure 1 Optimization of ESC culture and injection procedures. A FACS profile of three core ESC transcription factors Nanog Oct4 and Sox2 in C57BL/6J ESC clone 1.4cultured in 2i medium (Blue). Red population represents the isotype control. B BFACS profile of three core ESC transcription factors Nanog Oct4 and Sox2 in FVB/n ESC clone 1.3 cultured in 2i medium (Blue). Red population represents the isotype control. C Three ESC injection procedures for C57BL/6J ESC clone 1.4 were evaluated on basis of chimeric contribution. Injecting 4“8 ESC per FVB/n morula followed by overnight culture in KSOM medium provided the best chimeras with nine mice showing 100% coat color contribution (entirely black). male female n.d. D Two ESC injection procedures for FVB/n ESC clone 1.3 were evaluated on basis of chimeric contribution. Blastocyst injections resulted in reasonable chimeras whereas ESC injections into morulae in combination with overnight culture improved chimerism with three out of four live borns showing 100% chimerism (entirely white). The 80% chimera was a runt and died before weaning. male female n.d. EF Efficiency of ESC injection procedures shown in (C) and (D) based on number of viable chimeras born compared to the total number of implanted embryos for C57BL/6J ESC clone 1.4 (E) and FVB/n ESC clone 1.3 (F). Note for both ESC clones fewer chimeras were observed relative to the total number of implanted embryos when comparing ESC injected morulae to ESC injected blastocysts. "
1
"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4“ it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and œposed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ”ie epigenetic modifications made by the parent in generating the sex cells”which alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using œsex or œgender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet “diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand women™s heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and women™s hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charit”universittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for women™s health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients™ and doctors™ behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals™ everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20“ sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients™ perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33“ the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·influenza and pneumonia ·suicide ·alzheimer™s disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·influenza and pneumonia ·type diabetes ·injuries ·alzheimer™s disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal women™s hearts develop less inflammation resulting in less fibrosis than men™s hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33“ an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced women™s risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv
0
"objectives 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 cancerss 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 open 202010e037719 101136bmjopen2020037719 0copen 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 cprd™s 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 org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 œmalignant neoplasms of urinary organ 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 open 202010e037719 101136bmjopen2020037719 0copen 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 open 202010e037719 101136bmjopen2020037719 0copen 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 organ 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 open 202010e037719 101136bmjopen2020037719 0copen 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 open 202010e037719 101136bmjopen2020037719 0copen 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 open 202010e037719 101136bmjopen2020037719 0celitnecrep ht“htrqi inademelitnecreprqi inadem ht“htelitnecrep ht“ht inademrqielitnecrep ht“htcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen 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 ““““““““““““““ˆ’““““““ 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 ht“ht““““““““““““““sarcn 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 open 202010e037719 101136bmjopen2020037719 0copen 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 open 202010e037719 101136bmjopen2020037719 0copen 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 open 202010e037719 101136bmjopen2020037719 0copen 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 open 202010e037719 101136bmjopen2020037719 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 should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprd™s recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncras™s recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu
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pancreatic cancer is a devastating malignancy with a 5year relative survival rate of only “ dependenton the geographical location surveyed [“] with these statistics exhibiting only modest improvementover the last four decades [“] the median survival postdiagnosis ranges from just “ months forlocally advanced disease and “ months for metastatic disease it was estimated by the world healthanisation that pancreatic cancer is currently the 7th leading cause of cancerrelated death being responsible for over deaths worldwide in with incidence increasing pancreatic cancerhas been predicted to be the third leading cause of cancerrelated death in the european union by and the second leading cause of cancerrelated death in the united states of america and germanyby several factors contribute to the poor survival of pancreatic cancer patients a current lack of reliablediagnostic markers that would enable early screening coupled with largely nonspecific symptoms ofdisease results in over of patients presenting with metastatic disease at diagnosis this subgroupof patients have limited therapeutic options and are thus typically administered palliative chemotherapyaimed at prolonging survival and reducing symptoms during endoflife care [“] moreover whilstapproximately “ of patients present with localised disease that is eligible for potentially curativesurgery disease recurs in over of patients postresection ultimately these factorsculminate in more than of patients diagnosed with pancreatic cancer succumbing to disease these harrowing statistics highlight that despite research efforts there remains a lack of understandingof the pathogenesis of disease which in turn limits the development of new therapeuticsreceived march revised july accepted august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211pancreatic ductal adenocarcinomapancreatic ductal adenocarcinoma is the predominant pancreaticmalignancypancreatic cancer can arise from either the endocrine or the exocrine region of the pancreas tumours arising fromthe exocrine compartment are termed pancreatic ductal adenocarcinoma pdac and account for over of allpancreatic cancers pdac develops via a stepwise progression from normal tissue through to invasive lesions which is associated withdistinct morphological characteristics [“] it has been proposed that this process begins with a phenomenontermed acinartoductal metaplasia adm which is a normal homeostatic mechanism whereby acinar cells transdifferentiate into a ductal phenotype in response to particular stimuli however if compounded by an oncogenic˜hit™ cells are pushed towards a pathogenic phenotype that develops into pancreatic intraepithelial neoplasia panin[“] disease progresses through preinvasive stages termed panin1a panin1b panin2 and panin3 priorto invasive pdac this progression is associated with increasing nuclear atypia whereby the nuclei are no longerpositioned basally and loss of normal architecture as cells become more papillary in nature with panin3 lesionsdemonstrating increased mitoses as disease progresses to pdac cells become invasive and breach the basement membrane growing through the extracellular matrix and metastasising to distant ans figure less common precursor lesions include intraductal papillary mucinous neoplasms ipmns and mucinous cysticneoplasms mcns that also develop through multistep processes whilst they share some common featureseach lesion is morphologically and genetically distinct in contrast with panins that form within small ducts ipmnsdevelop within the primary or secondary branches of the main pancreatic duct whilst mcns lack ductal involvement an ‚ammatory tumour microenvironment contributes to pdacpathogenesisan archetypal feature of pdac is the development of extensive stromal networks within the tumour microenvironment tme that can account for up to of the total tumour volume [“] this unique characteristic drives theinflammatory nature of pdac that contributes to its aggressive phenotype desmoplasia is driven by pancreaticstellate cells pscs and cancerassociated fibroblasts cafs that upon activation produce a range of extracellularmatrix ecm components such as collagen laminin and fibronectin which in turn form a physical barrier preventing the penetration of therapeutics [“] though pscs and cafs have been shown to support cancer metastasis and drug resistance they interact with cancer cells in a bidirectional manner with each promoting the survivalgrowth and proliferation of the other [“] quiescent fibroblasts are able to differentiate into two unique subtypes termed myofibroblastic cafs mycafs and inflammatory cafs icafs these two subtypes are distinct whereby mycafs express high levels of αsmooth muscle actin αsma and are located adjacent to cancercells while icafs express low levels of αsma and instead secrete high levels of inflammatory mediators including il6 and are distributed distant from cancer cells within desmoplastic tumour regions broadly mycafsappear to have roles in epithelialtomesenchymal transition emt and ecm remodelling whilst icafs appear tobe involved in inflammation and ecm deposition a third less abundant subtype termed antigenpresentingcafs apcafs has more recently been defined these cells express low levels of both αsma and il6 andinstead express high levels of major histocompatibility complex class ii mhcii and related genes as such allthree subtypes are transcriptionally and functionally distinctthe wider tme contains a plethora of other cell types including endothelial cells tumourassociated macrophagestams and neutrophils tans mast cells regulatory tcells myeloidderived suppressor cells mdscs dendriticcells natural killer nk cells and nerve cells interactions between various cells within the tme can driveeither proor antitumorigenic functions of others for example cancer cells can induce pscs to secrete inflammatorycytokines that drive immune cells towards an immunosuppressive phenotype and also form a positive feedback loopby increasing the tumorigenic potential of cancer cells the ecm itself has also been suggested to modifypsc behaviour in particular that ecm stiffness promotes the mycaf phenotype indicated by increased αsmaexpression this results in substantial complexity that ultimately determines tumour phenotype the components of the microenvironment modify tumour behaviour through the production of cytokines growthfactors and other signalling molecules that predominantly drive a proinflammatory and immunosuppressive program that enhances pdac tumour growth and progression [“] figure the ability of the tme toinhibit therapeutic efficacy through both molecular mechanisms and physical fibrotic barriers contributes to the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211figure our current understanding of the contribution of il6 family cytokines to panin and pdac developmentpreinvasive panin lesions develop from normal ductal epithelia through panin stages 1a 1b and to stage invasive andormetastatic pdac this process is associated with acinartoductal metaplasia adm early in disease combined with an accumulation of oncogenic mutations most common mutations are indicated a number of cells within the tumour microenvironment havebeen shown to secrete il6 family cytokines which in turn results in the activation of a protumorigenic signalling cascade a betterunderstanding of the relationship between each of these cells within the tumour microenvironment may reveal new therapeuticopportunities to manage cancer progressionintrinsic resistance of disease thus dual targeting of cancer cells and the tme may be required to induce afavourable therapeutic response although this poses a signficant scientific and clinical challenge [“]molecular basis of pathogenesispdac development is associated with accumulation of mutationsthe progression of tumorigenesis through panin and pdac stages is associated with the stepwise accumulation ofspecific genetic mutations that drive malignant transformation the most frequent genetic alteration is an activatingkras point mutation codon that occurs early on in tumour development and is detected in over ofpdac tumours [“] mutations in kras have been shown to drive development of precursor panin lesions andwhen combined with an appropriate tumour suppressor mutation these lesions progress to invasive or metastaticpdac figure patient tumours harbouring wildtype wt kras often carry activating mutations indownstream effector molecules such as braf or pik3ca the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211inactivation of a range of tumour suppressor proteins is also common including mutations in tp53 cdkn2aand smad4 in approximately and of tumours respectively whilst each mutation has uniquemechanistic outcomes all three proteins are either directly or indirectly involved in the regulation of the g1s cellcycle checkpoint analysis of patient tumours indicates that two or more of these mutations often occur together withcdkn2a mutation being combined with either tp53 smad4 or both usually in the background of kras mutation this suggests that by disrupting this checkpoint cancer cells are able to overcome inhibitory mechanismsallowing continued progression to invasive diseaseunbiased sequencing efforts have also enabled identification of low prevalence pdac mutations observed in lessthan of cases these include mutations in genes involved in chromatin modification kdm6a dnadamage repair atm and other tumourrelated processes such as growth tgfbr1 or tgfbr2 furthermore it is important to note that technical advances are continuously uncovering epigenetic mechanisms thatfurther modulate the pdac transcriptional landscape and ultimately influence disease heterogeneity and tumourprogression molecular subtypesthe pdac epithelial compartment is typically divided into two subtypes including a classical subtype exhibiting anepitheliallike expression profile and a squamous or mesenchymallike subtype an additional third exocrinesubtype is outlined in some analyses and is characterised by a gene expression profile related to digestive enzymeproduction the classical or epithelial subtype has also been further divided into a pancreatic progenitor andan immunogenic subtype whereby the immunogenic subtype is distinguished by significant immune infiltration andassociated gene programmes though there is no consensus on which classification system will allow the mostvaluable stratification of patients the mesenchymal subtype is invariably associated with a poor prognosis the stromal compartment has also been classified into either normal or activated subtypes reflecting the proandantitumorigenic capabilities of the tme with the activated subtype associated with reduced survival this isparticularly valuable as the extensive heterogeneity of pdac complicates clinically relevant stratification of patientsthus the identification of molecularly unique subtypes may enable development of tailored therapeutic regimensthat will provide improved clinical outcomescurrent treatment optionsregardless of disease stage at time of diagnosis patients have relatively limited treatment options for the majorityof patients disease will be locally advanced or metastatic disqualifying them from undergoing potentially curativesurgery in these cases patients are typically offered chemotherapy with palliative intent [“]surgery provides the only potentially curative treatmentsurgical resection remains the only potentially curative treatment option due to minimal efficacy of standardofcarechemotherapy and radiotherapy due to its aggressive nature the majority of patients present to clinic with locallyadvanced or metastatic disease with only “ of patients presenting with localised tumours that are eligiblefor surgical resection even for those able to undergo surgical intervention over of patients relapsepostresection with median survival improving to months and 5year relative survival rate increasingmodestly to “ the use of neoadjuvant therapy is generally reserved for borderline resectable disease inan effort to enable patients to become eligible for surgery however a range of recent trials have shown improved clinical outcomes including overall survival for neoadjuvant treatment of patients with resectable tumours following surgical resection patients are typically treated with adjuvant gemcitabinebased chemotherapy although a recent study showed improved diseasefree survival and overall survival with a modified folfirinoxtherapy combination of oxaliplatin irinotecan leucovorin and fluorouracil radiotherapy provides variable clinical outcomewhilst the use of radiotherapy and chemoradiotherapy combination chemo and radiotherapy in the neoadjuvantand adjuvant settings have been investigated there remains a lack of consensus regarding therapeutic benefit this is due to issues such as insufficient radiation dose and low participant numbers as well as low uptake of moderntechniques in the neoadjuvant setting preliminary studies reported reduced lymph node positivity and rates oflocal recurrence for chemoradiotherapy compared to surgery with adjuvant chemotherapy however the useof radiotherapy in the palliative setting was reported to modestly reduce overall survival more recent studiesusing ablative doses of radiation have shown a survival benefit highlighting that technological advancements mayprovide an avenue for improved clinical outcomes following radiotherapy these contrasting results highlight the the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211need to determine which subset of patients may benefit from the inclusion of radiotherapy approaches in standardtreatment regimenschemotherapy remains the cornerstone of treatmentdespite modest improvements in overall survival palliative chemotherapy remains the standard treatment optionfor patients with locally advanced or metastatic disease gemcitabine monotherapy has been the mainstay treatmentfor pancreatic cancer since when it was demonstrated to improve median survival by just over month compared with fluorouracil within the last decade there have been some further improvements in clinical outcomewith combination chemotherapies gemcitabinenabpaclitaxel and folfirinox providing median overall survivalbenefits of and months respectively compared with gemcitabine alone although folfirinox treatment resulted in a lower percentage of patients experiencing reduced quality of life it also had increased toxicity andadverse events thus preventing its administration to patients with multiple comorbidities therapeutic resistance remains a significant barrier to patient survivaldespite advances in chemotherapeutic options treatment efficacy and patient prognosis remain poor due to the inherent therapeutic resistance of pancreatic cancer it has been proposed that this drug resistance may be driven by thetme including changes to cytokine signalling and metabolic pathways [“] this intrinsic resistance is demonstrated by patients experiencing similar overall survival for chemotherapy treatment “ months compared withbest supportive care “ months which encompasses the use of palliative surgery psychological support painmanagement and other methods of symptom control whilst a range of targeted treatments such as egfr orcheckpoint inhibitors have been trialled with or without chemotherapy they have shown limited success [“]emerging roles for the il6 family of cytokines in pdaccytokines are soluble molecular messengers that enable efficient communication between a range of cell types andhave been recognised to be major contributors to the growth and metastasis of cancers [“] in pancreatic cancer cytokines mediate signalling between cancer cells and the cells of the tme including pscs cafs endothelialcells and a range of immune cells including macrophages mast cells neutrophils and regulatory tcells [“]it is the specific signalling pathways active within this community of cells that dictates the balance of pro andantitumorigenic functions the il6 family of cytokinesthe interleukin il6 family of cytokines includes il6 il11 leukaemia inhibitory factor lif oncostatin mosm ciliary neurotrophic factor cntf cardiotrophin1 ct1 cardiotrophinlike cytokine clc neuropoietin np il27 and il31 [“] these cytokines are grouped as they share structural similarity forming a fourαhelical bundle termed helices ad with an upupdowndown topology il6 and il11 utilise a hexameric signalling complex consisting of two molecules each of the cytokine αreceptoreither il6r or il11r respectively and βreceptor glycoprotein gp130 [“] il6 and il11 are ableto signal via two distinct mechanisms termed classic and transsignalling classic signalling involves the formation of a complex including membranebound il6r or il11r with gp130 and the respective cytokine converselytranssignalling utilises soluble il6r or il11r molecules which are able to form a signalling complex with gp130and the respective cytokine [“] in this way classic signalling relies on the responding cell™s intrinsic expressionof il6r or il11r whilst transsignalling is able to activate any cell expressing gp130 lif osm il27 and il31 signal through trimeric complexes with a single cytokine molecule engagingthe respective receptor lifr osmr il27r wsx1 or il31r and either gp130 or osmr for il31[“] cntf ct1 clc and np form tetrameric signalling complexes composed of one cytokinemolecule one lifr one cntfr and one gp130 receptor in each case the active signalling complex consists of two chains that are signalling competent with a combination of either gp130 lifr osmr il27r or il31r the requirement for multiple receptor subunits means that although gp130 is ubiquitously expressed the expression of other receptor subunits dictates the ability for any given cell to respond to cytokine as signalling initiationrequires the presence of cytokine and a compatible receptor complex figure 2asignalling complex assembly leads to transphosphorylation and activation of receptorassociated janus tyrosinekinases jaks largely jak1 and to a lesser extent jak2 and tyk2 in the case of gp130mediated signalling this results in phosphorylation of the cytoplasmic domain of gp130 at tyrosine y and phosphotyrosine py and of gp130 provide docking sites for signal transducer and the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211figure il6 family cytokine signalling pathwaya schematic representation of the stepwise binding process for the il6 family members with il6 as an example the site interaction involves cytokine binding to the respective receptor with the site interaction generally between the cytokine and thecommon gp130 receptor chain finally site interactions involve formation of the final active signalling complex in this case formation of the il6il6rgp130 hexameric complex b general outline of the il6 family cytokine signalling pathway formation ofan active hexameric complex leads to activation of jaks with subsequent activation of the stat3 mapk and pi3k pathways leftthis results in upregulation of the negative regulator socs3 as well as a range of inflammatory and protumorigenic moleculesthe pathway is inhibited by socs3 pias3 and ptps via dephosphorylation ubiquitinmediated proteasomal degradation andsumoylation right the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211activator of transcription stat molecules leading to their subsequent phosphorylation by jak1 and formation ofactive stat dimers [“] phosphorylated stat pstat dimers then translocate to the nucleus and modulatetarget gene expression including upregulation of a range of genes involved in inflammatory and protumorigenicpathways [“] figure 2b broadly these stat3regulated genes can be categorised into pathways associatedwith inhibition of apoptosis increased cell proliferation modulation of immunity and inflammation increased angiogenesis and increased invasive and metastatic potential [“]although jakstat signalling is the predominant pathway activated downstream of il6 family cytokines themitogenactivated protein kinase mapk and phosphoinositide 3kinase pi3k pathways can also be activated the mapk pathway has been suggested to be activated by a src homology domain 2containing phosphatase shp2mediated mechanism whereby shp2 is recruited to py759 on gp130 allowing jakmediated phosphorylation of shp2 this promotes association with the adaptor protein growth factor receptor bound protein grb2 leading to activation of the gprotein ras via son of sevenless sos with a subsequent phosphorylationcascade including raf mek and erk12 activity following this a mapkdependent phosphorylationevent leads to the recruitment of grb2associated binding protein gab1 to the plasma membrane where gab1 issuggested to act as a scaffold or adaptor protein to allow binding of pi3k and shp2 leading to activation of the pi3kand mapk pathways respectively figure 2bthe suppressor of cytokine signalling socs3 is largely responsible for regulation of signalling and is directlyupregulated by stat3 socs3 contains an sh2 domain allowing it to bind to py residues within the gp130receptor with preferential binding to y759 once bound socs3 recruits an e3 ubiquitin ligasecomplex containing cullin5 rbx2 and adaptors elongin b and c via its socs box domain this complexubiquitinates the gp130 receptor inducing its internalisation and targeting it for proteasomal degradation [“]and is also able to ubiquitinate jak2 in vitro socs3 also mediates direct inhibition of the kinase activityof jak12 via its kinase inhibitory region [“] thus socs3 is able to downregulate il6 family cytokinesignalling pathways through two distinct mechanismsthe phosphotyrosine phosphatases ptps and protein inhibitors of activated stats piass also limit the strengthand duration of cytokine signalling a range of ptps including shp2 are responsible for dephosphorylatingtyrosine phosphorylated substrates including jaks stats and other shp2 molecules pias3 preferentially binds pstat3 and inhibits activity either by preventing stat3 interaction with dna by recruiting transcriptional repressors to stat3 target genes or by sumoylating stat3 to prevent its activity figure 2binterleukin in pdacelevation of serum il6 is a negative prognostic marker in human pdacserum il6 levels were increased in pdac patients compared with healthy patients [“] or those withchronic pancreatitis and were also increased in patients with metastatic pdac compared to thosewith locally advanced disease [“] moreover elevated serum il6 positively correlated with increased diseaseburden weight losscachexia and metastasis [““] however there are conflicting observations inthe literature regarding il6 and cachexia although increased serum il6 levels correlate with increased disease stage and in metastatic patients correlates with poor overall survival as such it has been suggestedthat il6 may be a superior marker for diagnostic and prognostic purposes compared with the standard creactiveprotein crp carcinoembryonic antigen cea and carbohydrate antigen ca199 markers il6 is expressed within the tmell6il6 was overexpressed in human pdac tumours in comparison with adjacent normal tissue whilstthis tumourspecific elevation has been correlated with reduced survival in some studies othersshowed no significant correlation with survival similar to the data available in the cancer genome atlastcga dataset for both il6 and il6r figure 3ab the tcga comprise aggregate sequencing data which doeshave limitations regarding interpretation of contributions of individual cell populations to disease outcome howeverit remains a widely used resource for exploratory investigations however overexpression of il6 has been observedat the mrna and protein level in the pancreata of pdac mice with il6 expression increasing with agewhich is indicative of disease stage in these models despite the presence of il6 in tumours primary human and commercial pancreatic cancer cell lines have been reported to exhibit variable expression levels of il6 and secreted cytokine albeit consistently higher than normal pancreatic ductal epithelial cells in an anoid model minimal il6 was expressed by pancreaticcancer cells pccs or pscs in monoculture however in coculture pccs expressed only il6ra whilst icafs expressedhigh levels of il6 with this activating stat3 within pccs icafs also demonstrate an upregulation of the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211figure il6 family cytokine expression in pdac patientsoverall survival for patients with high top quartile and low bottom quartile level expression of a il6 b il6r c il11 d il11re lif f osm g cntf h ctf1 ct1 i clcf1 clc and j il27 n per group data and graphs obtained fromoncolnc using data from the cancer genome atlas tcga statistical significance determined by mantelcox logranktest the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211the jakstat pathway with expression of il6 being dramatically increased in vitro when incubated with pcc conditioned media indicating that soluble factors trigger il6 production more recently pccderived il1αhas been shown to induce autocrine lif secretion and thereby promote the icaf phenotype including activation ofthe jakstat signalling pathway and il6 production in addition tams have been identified as producers of il6 in pancreatic cancer by correlative immunohistochemistry and expression analysis of isolated cell populations production of il6 by tams was shownto influence tumour development via bonemarrow chimeras as mice reconstituted with il6 knockout ko il6myeloid cells developed lowgrade panins whilst those reconstituted with il6 wt cells developed panin3 lesions il6 is a driver of pdac pathogenesisboth in vitro and in vivo studies suggest that the presence of il6 in the tme can drive activation of stat3 with il6 inhibition reducing stat3 phosphorylation this il6stat3 program has been proposed tobe a driver of pdac pathogenesis by enhancing tumour initiation and progression angiogenesis regulation of cytokine expression and immune cell behaviour resistance to apoptosis and promotion of metastasis [“] in aninducible krasdriven mouse model genetic deletion of il6 resulted in a reduction of adm and panin formationwhen kras mutation was initiated embryonically compared with controls suggesting a role for il6 in tumour initiation this was also observed in a constitutive kras mutant model where genetic deletion of il6 preventedtumour initiation in vivo with a reduction in the number of panin and lesions interestingly oncogenickras and hypoxic conditions both features of pdac tumours were shown to induce il6 production perhaps representing a feedforward pathway enhancing tumorigenesis however il6 is notabsolutely required for panin formation as induction of kras mutation at weeks of age in conjunction with anexperimental pancreatitis model drove formation of panin lesions that were not significantly different between il6wt and ko mice il6 mice exhibited reduced tumour progression with decreased proliferative capacity of both cancer and stromal cells enabling regression of precursor lesions furthermore this inhibition of tumour progression by il6deletion was due at least in part to the reversal of adm with ductal cells reverting to an acinarlike phenotype increased apoptosis of cancer and stromal cells was also shown to contribute to this reduced tumour progression as demonstrated by appropriate immunohistochemical analyses with upregulation of proapoptotic anddownregulation of antiapoptotic bcl2 family members this is mirrored by in vitro data whereby il6 stimulation increased the expression of antiapoptotic bcl2bcl2 and bcl2l1bclxl with blockade of il6signalling or stat3 activation inducing apoptosis collectively these data suggest that whilst il6 contributes it is not required for pdac initiation and progressionthe process of angiogenesis supports tumour growth and progression by enabling adequate blood supply whichis enhanced by il6 signalling upon il6 stimulation pdac cell lines upregulate key angiogenic factors such asvascular endothelial growth factor vegfvegf and neurophilin1 nrp1nrp1 with significant correlation observed between the expression of il6r and vegf on human pdac sections il6inducedupregulation of vegf correlated with a growth advantage in pccs with both features inhibited by treatment witha jak2 inhibitor another facet of the protumorigenic effects of il6 is the regulation of cytokine expression that enables modulationof the immune system in particular it has been shown that il6 is able to upregulate a type cytokine profile invitro that may inhibit antitumour immunity in disease il6 suppressed the differentiation of human cd14cells into dendritic cells dcs in vitro whilst combination treatment with il6 and granulocyte colonystimulatingfactor gcsf inhibited the ability of dcs to respond to alloantigen a process that is required for dc maturationand antigen presentation where these effects were reversed by blockade of il6 andor gcsf il6 has alsobeen implicated in driving increased apoptosis of type i conventional dcs cdc1s leading to cdc1 dysfunctionearly in tumorigenesis and thereby decreased cd8 tcell activation this notion is further supported by invivo studies whereby genetic deletion of il6 in a krasdriven pdac mouse model exhibited a significant decreasein the percentage of intratumoral cancerpromoting macrophages and mdscs utilising primary human pscsmdsc differentiation was shown to be driven by pscderived il6 in a stat3dependent manner the resultantmdscs were able to suppress tcell proliferation suggesting a role for il6 in promoting an immunosuppressivetme correlative analysis indicates that il6 through the generation of metabolic stress indirectly causes a reductionin the percentage of intratumoral natural killer nk cells cd4 and cd8 tcells in precachectic and cachecticmice this effect was coupled to a reduction in the expression of an array of genes involved in immune cell the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211recruitment and tcell effector function indicating that il6 is able to directly and indirectly modulate immuneregulation to enhance tumorigenesisemt migration and invasion are prerequisite abilities that are required for tumour metastasis stimulation of pccswith il6 modulated the expression of a range of proteins that drive emt and migration including upregulationof snai1snai1 snail snai2 slug cdh2 ncadherin vim vimentin fn1 fibronectin col1a1 collagen and twist2 and downregulation of cdh1cdh1 ecadherin with these effects mitigated by il6 orstat3 inhibition il6treated pccs and preinvasive panins exhibited morphological cha
0
" 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 efficacy 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 IntroductionInflammatory 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 fistulas 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Ž 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 ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot 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 financial 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 tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy 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 efficacy 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 findings 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 reflecting 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 aspecificity 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 first of onset areassociated with a poor prognosis at the first 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 efficient at reflecting disease activity []Some studies have also investigated the efficacy 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 efficacy 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 efficacy 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 ˆ’ °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 specificity 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 first evidence of the efficacy 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 first 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 confirmed and complemented the findings 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 specificity 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 specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity 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 specificity 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 specificity [] Caviglia et al in their study reported a sensitivity of and a specificity 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 specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity 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 findings it seems that fecal calprotectin has no ideal sensitivity and specificity 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 efficacy 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 andspecificity 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 andspecificity 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 findings 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 firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first 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 findings 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 SpecificityReferences[][][][][]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 specificity 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 findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity 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 diseaseidentification 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 modifiedBaron 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 specificity 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¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 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 indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity 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 specificity 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 specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed 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 specificity 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 thedifficulty 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 isRobert™s 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 Robert™s scoring system [] Theede et al also used themodified 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 Specificity 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 specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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" a critical barrier to improving the quality of endoflife eol cancer care is our lack of understanding of themechanisms underlying variation in eol treatment intensity this study aims to fill this gap by identifying anizational and provider practice norms at major us cancer centers and how these norms influence providerdecision making heuristics and patient expectations for eol care particularly for minority patients with advanced cancermethods this is a multicenter qualitative case study at six national comprehensive cancer network nccn andnational cancer institute nci comprehensive cancer centers we will theoretically sample centers based upon nationalquality forum nqf endorsed eol quality metrics and demographics to ensure heterogeneity in eol intensity andregion a multidisciplinary team of clinician and nonclinician researchers will conduct direct observations semistructuredinterviews and artifact collection participants will include cancer center and clinical service line administrators providers from medical surgical and radiation oncology palliative or supportive care intensive care hospital medicineand emergency medicine who see patients with cancer and have high clinical practice volume or high local influenceprovider interviews and observations and adult patients with metastatic solid tumors and whom the providerwould not be surprised if they died in the next months and their caregivers patient and caregiver interviewsleadership interviews will probe about eol institutional norms and anization we will observe inpatient andoutpatient care for two weeks provider interviews will use vignettes to probe explicit and implicit motivations fortreatment choices semistructured interviews with patients near eol or their family members and caregivers willexplore past current and future decisions related to their cancer care we will import transcribed field notes andinterviews into dedoose software for qualitative data management and analysis and we will develop and apply adeductive and inductive codebook to the datadiscussion this study aims to improve our understanding of anizational and provider practice norms pertinent toeol care in us cancer centers this research will ultimately be used to inform a provideroriented intervention toimprove eol care for racial and ethnic minority patients with advanced cancertrial registration clinicaltrialsgov nct03780816 december keywords endoflife norms heuristics cancer minority health correspondence amberbarnatodartmouthedu2the dartmouth institute for health policy and clinical practice geisel schoolof medicine dartmouth college lebanon nh usa5department of medicine geisel school of medicine hanover nh 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 0cknutzen bmc palliative care page of the national academy of medicine has identified increasingly aggressive burdensome and expensive endoflife eol treatment as a major public health problem the american society of clinical oncology andnational quality forum nqf define aggressive burdensome and expensive eol treatment in cancer as thereceipt of chemotherapy in the last days of life nqf intensive care unit icu admission in the last days of life nqf and non nqf orlate nqf hospice referral such treatmentadversely affects patient quality of life quality of dyingand caregiver bereavement outcomes [“] minoritiesare more likely to receive such eol treatment [“]potentially in disproportion to their preferences [ ]despite attention focused upon integrating early palliative care into advanced cancer treatment [“] icuadmission in the last days of life and late hospicereferral have been secularly increasing yet not allanizations are equal cancer centers vary by morethan twofold in these eol intensity measures [ ]these variations cannot be explained by structural characteristics or casemix since centers serving ahigher proportion of minority patients have systematically higher eol intensity than centers serving a higherproportion of white patients these variations inpractice patterns may contribute to racial disparities inburdensome treatment near death moreover despiteefforts to attribute such variation to differences in patient preferences rather regionlevel analyses suggest that the impact of thesepreferences on variation are likely very small [ ]than racial disparitiesin local anizational and providerwe posit that a critical barrier to improving thequality of eol cancer care in the us “ and amongminorities in particular “ is our lack of understandingregarding the mechanisms underlying cancer centervariation in eol treatment intensity the overarching hypothesis driving this study is that differencessocialnorms “ rules about which there is at least some degree of consensus enforced through social sanctions “ are a key mechanism underlying this variationwe base this hypothesis on our preliminary work attwo us academic medical center hospitals at oppositeextremes of eol treatmentintensity demonstratingmarked differencesicu and lifesustaining treatment decision making these normswere found to directly and indirectly via influencing patient and family treatment expectationsand provider decision making heuristics affect treatment decisions for minority and nonminority patients with advanced cancer norms are fruitfulforstudy because once understood they are potentiallymalleableandleadership effortsthrough explicitin norms ofimplementation of new forms of positive and negativesanctions via social marketing interventions our study aims to study mechanisms underlying cancer center variation in eol treatment intensity amongminority and nonminority patients using a qualitativecase study design and has two objectives first we willidentify the local anizational and provider practicenorms that influence decisions about laterline chemotherapy hospice and icu use among minorities withadvanced cancer at major us cancer centers second wewill assess the influence of these norms on patient andfamily expectations and provider decision making heuristics for laterline chemotherapy hospice and icu useamong minorities with advanced cancer at major uscancer centers below we describe our qualitative studydesign approach to meet our study objectivesrecommendationsmethodsdesignthe design of the studywe chose a qualitative case study design at six sites toidentify local anizational and provider practice normsthat influence variation in eol treatment intensity particularly for minority patients based on medicareclaims data analyses we will recruit of the ncinccn designated cancer centers serving at least african american advanced cancer patients webased our sample size on recent literature related tosample size sufficiencyto reachmultisite data saturation and qualitative research expertise of our study team first our study is guided by atheory based on previous research and uses data frommultiple sources to test and crosscheck for confirmingor disconfirming evidence of our theory a necessarycomponent of ensuring data sufficiency [ ] inaddition conducting qualitative case studies at six siteseach of which will include over interviews plus multiple observations will produce finegrained and rich descriptive analysis to generate and compare theoreticalinsights across sites as well as across stakeholders egproviders patients within sites [“] our targetnumbers for interviews and observations are well withinrecommendationsandsaturation given our well defined study aim lastlyresearch suggests that qualitative research expertise including the quality and depth of interview process is animportant criterion to consider for assessing sample size our site visit team has over years of collectiveexperience conducting qualitative research in healthcaretopics and settings ensuring a rigorous and thoroughprocess at each of the six sitesreaching dataadequacyforwe willtarget national comprehensive cancernetwork nccn and national cancer institute ncicomprehensive cancer centers for our study becausethey set national standards for high quality cancer care 0cknutzen bmc palliative care page of we will engage up to six sites serving a high proportionof african american patients ranging in eol care intensity and theoretically sampled to maximize our ability to compare and contrast anizational and providerpractice norms related to eol care we defined highproportion of minority patients as we measuredeol care intensity based upon riskadjusted metrics ofeol quality using medicare feeforservice claimsdata receipt of chemotherapy in the last days of lifenqf intensive care unit icu admission in thelast days of life nqf and non nqf or late nqf hospice referral our approach tocalculating these eol quality metrics has been publishedelsewhere given the multivariable nature of thesemetrics we use data visualization to purposively selectsites for case study that maximize potential heterogeneity in practice patterns following the principles ofpositive deviance sampling[ ] we will samplehigher versus lower eol quality sites in a ratio of we will employ qualitative case study researchmethods in this study including inpatient observationprocedures and provider patient and leadership semistructured interviews that have been previously developed and piloted [ “] we will augment thesemethods with outpatient and tumor board observationprocedures which we developed and tested at a nonstudy ncidesignated cancer center serving a whiterural population we williteratively revise all datacollection procedures based on researcher experiencesand thematic insights following each sampled case studysite visitqualitative data collectionthe study team will collect types of data field notesfrom direct observation of inpatient and outpatient cancer care and cancer tumor boards transcribed audiorecorded semistructured interviews with cancer centerleadership providers and patients family members andcaregivers and artifacts table we will link all datafrom observations using a unique identification idnumber we will use a data collection form for field observations to capture provider and patient demographicinformation as well as location time and individualspresent at the encounter in addition to relevant clinicaldata observers will note sociolinguistic dimensionssuch as turn taking tone affect body positioning andeye contact artifacts collected during the site visit willinclude workflows marketinginformational materialsorientation guidelines quality reporting and communication documents used in the cancer centersemistructured interview guides for site leadershipfocus on institutional norms including resources programs and policies related to eol care and outcomesas well as sitespecific workflows and scheduling logisticsin preparation for site visits cognitive mental modelssemistructured interview guides for providers whichtable data collection rationaledata collection methoddirect observationrationaleto learn about eol care for minority patients with advanced cancer specifically how it is influenced by anizational and provider practice norms provider decision making heuristics patient and family expectationssemistructured interviewsleadershipto probe anizationlevel norms including resources programs and policies sitespecific workflows and scheduling logisticsprovidersto explore individuallevel motivations decision heuristics andor rationalizations unconscious beliefs and assumptions that structure advanced cancer decision making using casevignettes to prime mental modelspatients family memberscaregiversto probe individuallevel preferences for cancer care past current and future decisions related to cancer careartifact collectionto learn how the anization standardizes workflows marketinginformational materials orientation guidelinesquality reporting and communication documents used in the cancer center and how this impacts local anizational and provider practice norms provider decision making heuristics patient and family expectations 0cknutzen bmc palliative care page of include eol case vignettes explore motivations decision heuristics andor rationalizations six eol vignettes were developed bya medical oncologistradiation oncologist and palliative care providers tohighlight key decision points common to outpatient orinpatient providers see table each vignette has oneversion with a photo of an african american patient andone with a photo of a white patient providers will viewone african american case and one white patient caseto facilitate mental models debriefing and uncover unconscious beliefs and assumptions related to race thatstructure advanced cancer decision making semistructured interview guides for patients family membersand caregivers probe past current and future decisionsrelated to their cancer caresite visit teams will consist of “ researchers we willidentify a sitespecific principal investigator pi at eachsite to help facilitate support from site leadership andidentify and recruit informants for pre sitevisit interviews and providers for sitevisit observation and interviews up to two months in advance of the site visit thestudy team will conduct leadership interviews by phoneincluding physician and nursing leaders outpatient oncology practice managers and key referral service lineleaders from palliativesupportive care hospital medicine and intensive care we will approach other siteleaders for interviews at the suggestion of the site piwhen necessaryleadership interviews will take placeduring and after site visitsone month in advance of the site visit the study teamwill recruit providers for observation in the inpatientand outpatient setting the observation schedule will involve one researcher assigned to each observed providerfor a halfday observation in outpatient clinics morningtable vignette summariessettingvignette number andpatient racespecialtyinpatient african american white african americaninpatient white african americanmedicaloncology white african american whiteradiationoncology african americansurgicaloncology white african american outpatient whitevignette summary and key questionsummary yearold man with metastatic gastric cancer he was living in a skilled nursing facilityafter a long hospitalization for infection he is now hospitalized with recurrent fever respiratorydistress and anxietykey question how to manage anxiety and respiratory distress in a patient with advanced cancerand high risk for shortterm deathsummary yearold woman with recurrent metastatic pancreatic cancer and mild dementia she isscheduled to start palliative chemotherapy next week she presents to the ed with decliningperformance status decreased appetite and abdominal pain her hospital evaluation demonstratespoor kidney function low blood pressure and rapid breathing “ all worrisome for rapid constitutionaldeclinekey question how to manage a patient with an aggressive cancer presenting to the emergencydepartment with multiple signs of constitutional declinesummary yearold man with advanced metastatic colon cancer he is married and lives at homewith his wife he presents to clinic with pain weight loss and signs of cancer progression he asksœdo you think the chemo is workingkey question how to answer patient questions about prognosis and next steps in treatment ofadvanced cancer with limited treatment optionssummary yearold man with a new diagnosis of metastatic renal cell carcinoma he presents withseizures brain metastases and lung metastases he is unmarried and without children his performancestatus is poor and he is not able to make his own health care decisions his eldest brother is his durablepower of attorney and asks œdoc what would you do if he was your brotherkey question how to approach surrogate decision making about management approach for a patientwith poor prognosissummary yearold man with newly diagnosed nonmetastatic lung cancer he has severe lungdisease and significant vascular disease from heavy smoking he is a poor surgical candidate hementions that the stress of his cancer diagnosis has caused him to drink alcohol more heavily thanusual and he is coughing up about “ tablespoons of bright red blood dailykey question how to approach a patient with a new diagnosis of a potentially curable cancer whenthere are a number of red flags that the patient may do poorly with surgical treatmentsummary yearold woman with a recent diagnosis of pancreas cancer she has been hospitalizedwith weight loss pain and declining activity her evaluation shows a œborderline resectable pancreaticcancer initial treatment would be chemotherapy or chemoradiation if she could tolerate this she hasbeen unable to eat or ambulate for the last five days due to poor appetite and performance statusshe says œi™m a fighter not a quitter and œwith jesus anything is possible she then asks œwhat comesnextkey question how to approach a patient who has a œtreatable diagnosis but who does not have theperformance status to tolerate treatment 0cknutzen bmc palliative care page of or afternoon session the emergency department byshift and inpatient setting by timing of daily service orconsult rounds as well as scheduled tumor boards andfamily meetings selection criteria for providers to observe and interview focus on maximizing our ability toassess provider norms for advanced cancer care withinthe particular institutional context that we will exploreduring leadership interviews provider selection criteriaincludes providers who manage patients with metastatic solid tumors ie we excluded leukemia lymphoma and bone marrow transplant providers and haveeither high volumes of patients andor high peerinfluence as perceived by the site pi or other key informants we seek to recruit medical radiation and surgical oncology providers as well as palliativesupportivecare providers who see cancer patients see table fortarget sampling frame we also seek to recruit providersfrom intensive care hospital medicine and emergencymedicine who care for acutely ill cancer patients wepresent an example observation schedule in fig wewill ask all providersrecruited for observation tocomplete an interview interviews with providers willoccur in person during or by phone after the site visitwe will digitally record all interviews and compensateall providers for participating in an interviewat least weeks prior to the site visit we will sendflyers about the study with photos of the study team tothe site pi who will facilitate posting of the flier in publicsettings such as waiting rooms clinic rooms infusionsuites and inpatient units the purpose of this flyer is toalert nonconsented individuals to our study purposeand to provide instructions for opting outduring patient care observation researchers will directly approach patients and their familycaregivers following introduction by the consented providerifpatients or their familycaregiver verbally consent to beinterviewed the study team member will obtain contactinformation to arrange for a phone interview at thepatient family member or caregiver™s convenience at alater date selection criteria for patient interviews includes adults aged years or older with metastatic solid tumor whom the provider would not besurprised if they died in the next months and seenby a consented provider we will seek to recruit equalnumbers of minority and nonminority patients we willdigitally record all interviews and compensate each participant for participatingtheoretical modelfindingsqualitative data analysiswe will use a qualitative and mixed methods data analysis platform dedoose to manage and analyze all transcribed field notesinterviews and artifacts and linkrelevant data to contextual information eg patient andprovider race site features sociocultural research consultants llc we will develop a codebook first deductively using ourin theliterature and priorresearch and then inductivelythrough an iterative process of close readings and discussion of the data in order to identify additional codesthree qualitative researchers will apply the codebook tothe data two who will divide and code all the data andone who will assess reliability of coding by reviewing asubset ofthree qualitative researchers will discuss differences in coding and resolveby consensus we will repeat the analysis process aftereach site visit to conduct constant comparative analysisregarding similarities and differences between and withinsites in support of study aims and after completionof each site visit the study team will develop a writtensummary of preliminary quantitative and qualitativefindings specific to the site which will then be sent to allparticipants from that site to assess initial validity of thesitespecific findings after completion of all site visitsand analysis of data we will send final study reports toparticipating sitesthe coded data alltable target sampling frame at each sitedata collection settingoutpatientmedical oncologysurgical oncologyradiation oncologysupportivepalliative careemergency medicineinpatienthospital medicineintensive caresupportivepalliative careoncology consultrigor and reproducibilityour research team is also conscious of conducting purposefully informed and respectful research on the cancerexperiences of racial and ethnic minorities and we havetaken steps to ensure scientific rigor of our approachand results through study design development and willcontinue to do so through data collection and data analysis based in a relatively nonracially diverse geographicregion our team n is comprised of racial andethnic minority researchers as such we seek to incorporate greater diversity of racial and ethnic knowledge aswell as disciplinary perspective through an external advisory board with deep topical expertise in cancer carepalliative care racial and ethnic health equity and socialnorms additionally to address potential researcher biasthe entire study team completed implicit bias trainingn“““““““““ 0cknutzen bmc palliative care page of fig mock onsite observation schedule researchers will ideally observe relevant outpatient clinics during week and inpatient servicesduring week researchers will go to tumor boards attended by consented providers as well as other relevant staff meetings eg fellowsmeetings researchers will observe providers during either am or pm blocks using the alternating daily block to dictate field notes and conductinterviews with providers and patients onsitefocused on unconscious biases related to attitudes aboutrace ethnicity cancer cancer treatment death anddying one researcher participating in data collectionwill remain blinded to sites™ eol treatment intensityclassification until data collection is completefinally we will employ multiple methods of triangulation to assure comprehensiveness and validity of datatwo to three researchers of a multidisciplinary team willparticipate in each site visit and an additional three researchers will conduct qualitative analysisto satisfyinvestigator triangulation method triangulation will include direct observation semistructured interviews andartifact collection we will achieve data triangulation byobserving and interviewing leadership personnel providers and patients family members and caregivers ateach site of various s specialties and diagnoses respectively qualitative analysis will use both deductive and inductive methodsto achieve theorytriangulationethics approval and consent to participatethe study has been approved by the dartmouth collegecommittee forthe protection of human subjectsstudy00031129 and is considered minimal risk allparticipating sites will waive independent irb approval in favor of acknowledging dartmouth™s irb rely on dartmouth™s irb via a smart irb reliance or conduct a local ethical review and approval we willobtain a waiver of informed consent for participant observation all providers will provide written electronicconsent for observation and interview and allinterviewed leadership patients and families will provide oralconsent for interview we have obtained a certificate ofconfidentiality from the national institutes of healthnih for this studystaffwe will not record any identifiable or personal information about providers patients family members caregivers orin field notes except demographicinformation a unique id number will link data fromobservations and interviewsincluding demographicdata to consented participants the key linking the idnumber and identifying information of the consentedparticipants will be maintained on a passwordprotectedserver only the research team will have access to thelinkage file all data collected on individuals will belinked to their id number alone we will audiorecordand transcribe all handwritten field notes without anyidentifiable information we will store all original fieldnotes in a locked filing cabinet and all transcripts on apasswordprotected server to which only the researchteam will have access we will give a discreet lapel pinto all providers staff patientsfamily members and 0cknutzen bmc palliative care page of caregivers who do not wish to be observed as advertisedby the informational flyers posted prior to the studyteam™s arrival at the site we will not document any individual wearing such a pin in field notes nor will weapproach them for an interviewwe will give all individuals participating in interviewsan information sheet prior to the interview and we willobtain informed consent verbally at the time of theinterview we will obtain informed consent verbally asmany of the interviews will be conducted by phone either before or after the site visit the process of obtaining verbal consent has been approved by the dartmouthcollege committee forthe protection of humansubjects we will record all interviews and later professionally transcribe them without any identifiable information we will store all recordings and transcripts on apasswordprotected server to which only the researchteam will have access additionally we consulted theguidelines for endoflife research putforth in theœmethods of researching end of life care morecareproject while designing the protocol for this study specifically we considered the risks egintervieweedistress and rewards eg potential therapeutic effectthat qualitative interviews may have for patients familymemberstheseprotocolsgivers while designingand carediscussionour study is the first comprehensive qualitative study oflocal anizational and provider norms at minorityserving nccn and ncidesignated comprehensive uscancer centers if the aims of this study are achieved weexpect to identify targets for institutional change at cancer centers with lower eol quality metric performancethe two main deliverables of this research will be knowledge regarding norms and their impact on eoldecision making at participating cancer centers and identification of potential members of a community research advisory board to oversee future institutionlevelinterventions aimed at improving eol care respectiveto the first deliverable participating cancer centers willreceive a customized report of our findings about theirown center following completion of our site visit aftercompletion of all site visits we will work with theamerican cancer society and participating cancercenters to identify local chapters ofthe americancancer society acs and provider medical societieseg county medical and nursing societies at which wecan discuss our findings and their implications for localpatients and providersrespective to the second deliverable we anticipate theopportunity to develop institutionlevelinterventionsaimed at eol care in the future norms are fruitful forstudy because once understood they are potentiallymalleable through explicit leadership efforts and implementation of new forms of positive and negativesanctions specifically social marketing “ the use ofmarketing principles to influence human behavior to improve health or benefit society “ is a promisingstrategy for changing norms interventionists have successfully applied the principles of social marketing tochange hiv risk behaviors [ ] and palliative careconsultation use integrating social marketing interventions in cancer centers with high intensity eol carecould have the effect of improving the quality and costof cancer care particularly for racial and ethnic minorities further by studying norms of decision makingamong groups of physicians this project will overcomethe limitation of past research which uniformly hasneglected this important issueabbreviationsnccn national comprehensive cancer network nci national cancerinstitute eol endoflife nqf national quality forum icu intensive careunit id identification pi principal investigator irb institutional reviewboard acs american cancer societyacknowledgementsthank you to inas kayhal for her assistance with the cluster analysis for sitesampling and associated visualizations and to garrett wasp for his input onthe clinical vignettesauthors™ contributionskek led coordination of the study participated in design of the protocolcontributed to instrument development and led preparation of themanuscript aeb led design and writing of the grant and protocolcontributed to instrument development and participated in preparation ofthe manuscript kes participated in design and writing of the grant andprotocol led instrument development and participated in preparation of themanuscript gfm and rb participated in design of the protocol contributedto instrument development and participated in preparation of themanuscript gab and nsk created the clinical vignettes contributed toinstrument development and participated in design of the protocol ssacontributed to instrument development and participated in design of theprotocol the authors read and approved the final manuscriptfundingthis research is funded by the american cancer society grant number rsg1801701cphps the funding body had no role in the design of the studyand will have no role in the collection analysis and interpretation of data orwriting of the manuscriptavailability of data and materialsendoflife metrics data can be found at httpswwwdartmouthatlasinteractiveappsendoflifecancercare qualitative data will be deidentifiedand made available to researchers through the ninrfunded palliative careresearch cooperative qualitative data repository after analyses in support ofthe primary aims are complete all materials and instruments developed forthis study are available by request of the authorsethics approval and consent to participatethis study has been approved by the dartmouth college committee for theprotection of human subjects study00031129 this study is consideredminimal riskconsent for publicationnot applicable 0cknutzen bmc palliative care page of competing interestsall authors declare no competing interest with respect to the researchauthorship or publication of this author details1department of behavioral social and health education sciences rollinsschool of public health emory university atlanta ga usa 2the dartmouthinstitute for health policy and clinical practice geisel school of medicinedartmouth college lebanon nh usa 3department of general internalmedicine boston medical center boston ma usa 4evidera pharmaceuticalproduct development bethesda md usa 5department of medicine geiselschool of medicine hanover nh usa 6norris cotton cancer center atdartmouthhitchcock medical center lebanon nh usareceived july accepted august refere
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Research letterthe COVID19An analysis of SARSCoV2 cellentry genes identifies the intestineand colorectal cancer assusceptible tissuesEditorSARSCoV2 is the causative agentforpandemicCOVID19 has necessitated rapidchangesin surgical practice andanisation through both the initialpeak and ongoing recovery period1SARSCoV2 infects cells by interacting with the host cellsurfaceprotein ACE2 and utilises TMPRSS2in viralspike protein priming tofacilitate cell entry Fig 1a2 WhilstCOVID19 is predominantly a respiratory disease approximately of patients have concurrent gastroinsymptoms3 SARSCoV2testinalRNA and live virus have been identified in stool from COVID19 patientsand SARSCoV2 readilyinfectsintestinal anoids46 Despite thesecircumstantial data gastrointestinaltransmission has not yet been formally confirmed Cancers commonlyexpress different genes from the tissueof origin and it is largely unexploredtumours can be infectedwhetherwith SARSCoV2 We soughttoexplore the expression of ACE2 andTMPRSS2 in large publicly available normaltissue and pancancerexpression data sets to understandwhether levels of these genes identifysusceptible tissuesAnalysis ofthe normaltissueGenotype Tissue Expression projectGTEx dataset showed high ACE2expression in the testis small intestinekidney heart thyroid and adipose tissue Fig S1a supporting informationTMPRSS2 levels were highest in theprostateintestinepancreas lung salivary gland kidneythyroid and liver Fig S1b supportinginformation Whilst initial analysissuggested only kidney and thyroidcoexpressed high levels of ACE2 andTMPRSS2 closer inspection of smallstomachsmall BJS Society LtdPublished by John Wiley Sons Ltdinformationintestinal and colonic samples revealedheterogeneity with a subpopulationalso coexpressing highlevels that was confirmed by Kmeansclustering Fig 1bc Fig S1cd supportingIn colonictissue high levels of both genes werefound in younger patients and greaterTMPRSS2 expression in femalesalthough neither factor defined thesubpopulation observed Fig S2ajsupporting information Biopsylocation and tissuecompartmentfrom colon samples were explored andhigher levels of both genes were foundin the mucosa and proximal colonalthough these factors also failed tofully define the high coexpressingsubpopulation Fig S2kmsupporting information These normalexpression data appear to identify aproportion of individuals who in thegastrointestinal tract express high levels of both genes known to be involvedin cell entry of SARSCoV2The Cancer Genome Atlas programTCGA is the largest pantumourcollection of genomicand transcriptomic sequencing data Havingidentified the heterogeneous geneexpression within the normal gastrointestinaltract we interrogatedTCGA and GTEx gene expressiondata to identify relative expressionof ACE2 and TMPRSS2 in tumourscompared to their tissue of originGenerally there was no correlationbetween high expression of ACE2and TPMRSS2 in normal tissue andhigh tumour expression Fig S3adsupporting information Howeverwe identified colorectal cancer asunique amongst human malignancies by coexpressing higher levels ofboth ACE2 and TMPRSS2 relativeto normal Fig 1de We exploredTCGA data to identify if molecularsubgroups of colorectal cancers specific mutations or other commonlycollectedcouldvariablesclinicaldefine tumours with varying expression of ACE2 and TMPRSS2 Therewas a subtle yet significanttrendfor higher ACE2 expression withyounger age but no association withsex Fig S4a supporting information Possession of a BRAF mutationwas found to predict lower levels oftumour ACE2 however TMPRSS2expression was unchanged Fig S4bcsupporting information StrikinglyACE2 levels were very low in tumourswith microsatellite instability MSIFig 1f Cumulatively these data identify aproportion of healthy individuals assusceptible to putative SARSCoV2intestinal infection and that patientswith colorectal cancer may be at evengreater risk of infection Further clinical studies are urgently required toexplore this mode of transmission ofCOVID19Conflicts of Interest and Sourcesof FundingThere are no conflicts of interestsScientistSJAB is supported by an AdvancedFellowshipCliniciangrantfrom Cancer Research UKC14094A27178 and core fundingfrom Wellcome and MRC to theWellcomeMRC Cambridge StemCell InstituteMahnaz DarvishDamavandi1 JamesLaycock2 Christopher Ward1 MilouS van Driel1 Mae A Goldgraben3and Simon JA Buczacki121WellcomeMRC Cambridge Stem CellInstitute University of CambridgePuddicombe Way Cambridge UK2Cambridge Colorectal Unit CambridgeUniversity Hospitals NHS Trust HillsRoad Cambridge UKand 3Department of Medical GeneticsUniversity of Cambridge Addenbrooke™sTreatment Centre Cambridge UKSCICambridgeSiBucz 101002bjs11911BJS 0cFig ACE2 and TMPRSS2 are expressed heterogeneously in the normal intestine and colorectal cancerResearch letterabdfcea Schematic demonstrating the mode of entry of SARSCoV2 into cells via interactions with the cell surface proteins ACE2 and TMPRSS2 b Scatterplot of the expression of ACE2 and TMPRSS2 across all colon GTEx samples Yellow circle highlights high coexpressing samples c Scatter plot of theexpression of ACE2 and TMPRSS2 across all small intestine GTEx samples Yellow circle highlights high coexpressing samples d Box and whisker plotof expression of ACE2 between normal large intestine Grey TCGA and GTEx and colon and rectal cancer Red TCGA samples Median IQROneway ANOVA P TPM Transcripts per million e Box and whisker plot of expression of TMPRSS2 between normal large intestineGrey TCGA and GTEx and colon and rectal cancer Red TCGA samples Median IQR Oneway ANOVA P TPM Transcripts permillion f Box and scatter plot of ACE2 expression levels from TCGA COADREAD data sets between tumour subtypes CIN chromosomal instabilityMSI microsatellite instability GS genome stable and POLE DNA polymerase epsilon Median IQR KruskalWallis p BJS Society LtdPublished by John Wiley Sons LtdwwwbjscoukBJS 0cResearch letter S¸reide K Hallet J Matthews JBSchnitzbauer AA Line PD Lai PBS Immediate and longterm impactof the COVID19 pandemic ondelivery of surgical services Br J Surg 101002bjs [Epub ahead of print] Hoffmann M KleineWeber HSchroeder S Kruger N Herrler TErichsen S SARSCoV2 CellEntry Depends on ACE2 andTMPRSS2 and Is Blocked by aClinically Proven Protease InhibitorCell “280e8 Yang L Tu L Implications ofgastrointestinal manifestations ofCOVID19 Lancet Gastroenterol Hepatol “ Wu Y Guo C Tang L Hong ZZhou J Dong X Prolongedpresence of SARSCoV2 viral RNA infaecal samples Lancet GastroenterolHepatol “ Wang W Xu Y Gao R Lu R Han KWu G Detection of SARSCoV2in Different Types of ClinicalSpecimens Jama “ Lamers MM Beumer J van der Vaart JKnoops K Puschhof J Breugem TI SARSCoV2 productivelyinfects human gut enterocytes Science 101126scienceabc1669 [Epub ahead of print]Supporting informationAdditional supporting informationcan be found online in theSupporting Information section atthe end of the BJS Society LtdPublished by John Wiley Sons LtdwwwbjscoukBJS 0c'
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tetrastigma hemsleyanum diels et gilg t hemsleyanum mostly known as œsan ye qing is a kind of folk plant because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource it mainly grows in the eastern central southern and south western provinces of china such as zhejiang jiangsu guangxi fujian and yunnan provinces peng and wang t hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models xu as an edible plant the leaves of t hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body sun while the aerial parts of t hemsleyanum developed as potential new traditional chinese medicine tcm preparations guo corresponding author ningbo research institute of zhejiang university ningbo zhejiang people™s republic of china email address px4142163com x peng 101016jjep2020113247 received may received in revised form july accepted august ofethnopharmacology2642021113247availableonline12august2020037887412020elsevierbvallrightsreserved 0ct ji abbreviations t hemsleyanum tetrastigma hemsleyanum diels et gilg tcm uplcesiqtofmsms ultra high performance liquid traditional chinese medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκb 5hydroxytryptamine norepinephrine dopamine prostaglandin e2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin mic gsh mda nfκb 5ht ne da pge2 mapk mitogenactivated protein kinase lps celegans caenorhabditis elegans tnfα il1 il6 il12p40 interleukin subunit p40 stnfr1 soluble tnf receptors il10 il1 il4 inos tlr4 md2 myd88 myeloid differentiation protein jnk gpt got alp sod interleukin interleukin interleukin inducible no synthase tolllike receptor myeloid differentiation factor2 cjun nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory the root tubers of t hemsleyanum are extensively used either alone or in combination with other herbal medicines in tcm clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain sun chen and guo therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal in february t hemsleyanum was awarded as the new œeight famous kinds of tcm in zhejiang province meant that it has become a key object of industrialization development of zhejiang™s dominant large varieties of medicinal materials in covid19 broke out and has caused more than deaths in china and infection cases have been reported in more than countries hua shi xuan fei mixture approval number of zhejiang medicine z20200026000 which composed of t hemsleyanum has been approved by zhejiang provincial drug administration for clinical treatment of covid19 futhermore the modern pharmacological studies had shown that t hemsleyanum also had effects of antiinflammatory ji antioxidant hossain antivirus ding antitumor lin antipyretic yang and wang antihepatic injury ma et al immunomodulatory xu antibacterial chen hypoglycemic ru 2018ab etc numerous reports have demonstrated that the biological activities of t hemsleyanum are attributed to its many chemical components fu wang has reported isolated alkaloids from the aerial parts of t hemsleyanum wang ru extracted a novel polysaccharide tdgp3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin a secretory immunoglobulin a epithelialmesenchymal transition alt ast ha ln tbili tp ifnÎ iga siga emt mmps matrix metalloproteinase timps matrixmetallo proteinase cytc cat gshpx glutathione peroxidase tregs tgf cox2 foxp3 pdl taoc ccl4 cef hvj vsv a f s1 s2 pef cff eaf baf cytochrome c catalase regulatory t cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast hemagglutinating virus of japan vesicular stomatitis virus alkalicontaining extract of t hemsleyanum ketonecontaining extract of t hemsleyanum crude extract of t hemsleyanum crude extract of t hemsleyanum petroleum ether extractions of t hemsleyanum ethanol extract chloroform extractions of t hemsleyanum ethanol extract ethyl acetate extractions of t hemsleyanum ethanol extract nbutanol extractions of t hemsleyanum ethanol extract t hemsleyanum with a molecular weight of — da by enzymolysisultrasonic assisted extraction method ru 2019ab large amounts of flavonoids were found in leaves aerial parts and root tubers of t hemsleyanum xu 2014ab deng yu in addition t hemsleyanum also contains a variety of functional components such as anic acids hu phenolic acids liu minerals fan amino acids fu etc in recent years wild resources of t hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments in it was listed in the preferentially protected crop germplasm resources of zhejiang province based on our team™s preliminary research peng peng 2016ab li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of t hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization materials and methods the available information about the traditional uses phytochemicals and pharmacological properties of t hemsleyanum was searched via web of science google scholar pubmed science direct china national knowledge infrastructure cnki and springer search using chinese or english as the retrieval languages the keywords used include t hemsleyanum root tubers of t hemsleyanum radix tetrastigma ofethnopharmacology26420211132472 0ct ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words all references were from experimental studies and published prior to april were reviewed all chemical structures were drawn using chemdraw pro software heatclearing were botanical characteristics t hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose it is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus the optimum ph is between and the root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter fig the epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section the stem of t hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate the leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base the flowers of t hemsleyanum are small yellow green and ovate the flowering stage of t hemsleyanum ranges from april to june and the fruit phase is normally from august to november when the flower withered it will form a small green round fruit with the size of millet when it is mature the fruit will turn from green to red the berries are spherical and soft spherical traditional uses t hemsleyanum belonging to the family vitaceae was firstly recorded in ben cao gang mu ming dynasty ad the aliases of sanyeqing include shi hou zi shi bao zi shi lao shu lan shan hu lei dan zi po shi zhu tu jing wan sou jia feng san ye dui golden wire hanging gourd golden bell golden wire hanging potato etc the root tubers or whole grass of t hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of tcm such as zhi wu ming shi tu kao qing dynasty wu jiangxi herbal medicine common folk herbal medicine in zhejiang all of these ancient works described the effects of toxicityremoving t dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women national compilation team of chinese herbal medicine in the tcm culture the properties of t hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional chinese medicine and zhong hua ben cao shanghai science and technology press the channel tropism was lung heart liver and kidney meridians decocting with water or mashing for external application are the traditional possess methods of t hemsleyanum considering its extensive traditional effects many prescriptions containing t hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies excitingly it has reported that jinlian disinfection drink containing san ye qing combined with interferon can treat covid19 he jinqi tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was wei moreover zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage iii primary liver cancer jiang and gong in addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites ji chemical compounds of themsleyanum the chemical constituents of t hemsleyanum have been widely investigated sun sun zeng xu 2014ab fu fan chen ding 2015a fig the aerial part a root tuber b and raw herb c of t hemsleyanum ofethnopharmacology26420211132473 0ct ji b ding a total of one hundred and fortytwo compounds have been isolated and identified from t hemsleyanum until now the information about compound name molecular weight compound formula detection method analysis sample is summarized in table flavonoids and their glycosides modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from t hemsleyanum lin zhang table to date fiftyone flavonoids and their glycosides have been extracted and identified from t hemsleyanum in this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on c3² and c4™ on the b ring of flavonoid aglycone at present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry uplcesiqtofms has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution our team used uplcesiqtofms to identify chemical constituents from the aerial part of t hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc sun according to the report liu total flavonoids of t hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase mapk and nuclear factorκb nfκb in lung tissue moreover the flavonoids of t hemsleyanum had the activity of antilung cancer wei luteolin a flavonoid found in t hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers muhammad it is certain that t hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs polysaccharide saccharide is another important active ingredient extracted from t hemsleyanum shao polysaccharide has great potential in clinical application because of its unique pharmacological activity however due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures guo table the prescriptions and traditional uses of t hemsleyanum in china prescriptions name qingteng fengshi qufengshi yaojiu main composition jiu traditional use t hemsleyanum parabarium chunianum tsiang zanthoxylum nitidum roxb dc t hemsleyanum deeringia amaranthoides lam merr blumea aromatica wall dc t hemsleyanum deeringia amaranthoides lam merr zanthoxylum nitidum roxb dc panax notoginseng burk fh chen t hemsleyanum gypsum lonicera japonica thunb houttuynia cordata thunb ophiopogon japonicus linn f kergawl t hemsleyanum t hemsleyanum lysimachia christinae hance imperata cylindrica citrus reticulata blanco t hemsleyanum ginsenoside astragalus propinquus schischkin t hemsleyanum nepeta cataria l lonicera japonica thunb saposhnikovia divaricata trucz schischk huatuo fengtongbao capsule sanyeqing gypsum decoction sanyeqing power zhonggan mixture jinqi tablet hua shi xuan fei mixture extracted the polysaccharides from roots of t hemsleyanum rtp1 rtp2 and rtp3 were successively found by protein precipitation and purification moreover further study indicated rtp31 was high purity polysaccharide with a molecular weight of kda and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively ru 2018ab extracted a polysaccharide thp from t hemsleyanum with the average molecular weight estimated as kda the results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of in ru 2019ab successfully extracted polysaccharide thdp3 from t hemsleyanum with molecular weight of kda which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of moreover tdgp3 mainly consists of †’4αdgalap1†’ †’4dgalp1†’ and †’4αdglcp1†’ residues as backbones and dmanp1†’ †’36dmanp1†’ and αdaraf1†’residues as branches phenolic acids phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring as a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects twenty three phenolic acids no52“ table have been reported in the aerial parts of t hemsleyanum such as caffeic acid chlorogenic acid 1ogalloyldglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid there were twentyone phenolic acids in the root tuber of t hemsleyanum some of which were the same as aerial parts alkaloids alkaloids are a group of basic anic compounds containing nitrogen that exist in nature alkaloids are stored in small quantities in t hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare wang fu extracted the aerial parts of t hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and treatment of joint pain wind cold dampness arthralgia treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture treatment of infantile hyperpyretic convulsion treatment of blood avalanche leucorrhea treatment of liver cancer treatment of malignant tumor treatment of covid19 usage oral administration “ ml once times a day oral administration ml once times a day oral administration capsules once times a day references ministerial standard ministerial standard ministerial standard one dose a day decoct twice in water and take it “ times after mixing oral administration oral administration ml once times a day oral administration capsules once times a day oral administration ml once times a day xu gao jiang and gong wei zhejiang provincial drug administration ofethnopharmacology26420211132474 0ct ji detection mode analysis parts of sample reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber sun sun zeng sun sun sun zeng zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun zeng sun zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun xu 2014b sun zeng sun zeng sun zeng zeng sun sun sun sun xu 2014b sun xu 2014b sun zeng sun xu 2014b sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun fu sun sun xu 2014b fan xu 2014b fan sun continued on next page uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms table chemical constituents isolated from the different parts of t hemsleyanum name flavonoids and their glycosides quercetin quercitrin quercetin3oglucoside quercetin3orutinoside quercetin3galactoside quercetin3oxylosylglucoside quercetin3oxylosylglucose7orhamnoside orientin orientin2²²orhamnoside isoorientin isoorientin2²²orhamnoside isoorientin cid0 ²²oxyloside vitexin vitexin2²²orhamnoside vitexin2²²oglucoside vitexin2²²oarabinoside isovitexin isovitexin2²²orhamnoside isovitexin2²²oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8cxylosyl6cglucoside apigenin6cαlarabinose8cdglucose eriodictyol eriodictyolohexoside i eriodictyolohexoside ii luteolin luteolin6 8dichexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3oneohesperidin kaempferol3orhamnoside kaempferol7orhamnose3oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3ocarfuran7orhamnosyl glucoside daidzein biochanin a procyanidin dimmer procyanidin b1 procyanidin b2 procyanidin trimer phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr13cnmr ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms ofethnopharmacology26420211132475 0ct ji table continued name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1ogalloyldglucose protocatechol glucoside epigallocatechin vanillic acid1ofuran celery glucosyl ester protocatechuic acid1ofuran celery glucosyl ester methoxyphenol1ofuran glycosyloglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid scid0 trolline fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid dimeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid terpenoids and steroids sitosterol daucosterol campesterol stigmasterol 6obenzoyl daucosterol ergosterol taraxerone taraxerol αamyrine pteroside z ganoderic acid h 3epipapyriferic acid oleanic acid saponins ginsenoside rh1 detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr lcms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms gcms tcl hnmr cnmr ms gcms gcms ir hnmr eims ir hnmr ms ir hnmr ms ir hnmr ms ir eims uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms hnmr cnmr ms analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber reference sun sun sun sun sun sun zeng sun xu 2014b zeng zeng zeng zeng xu 2014b xu 2014b chen fu fu fu fu fu fu fu fu fu fu sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun chen ding sun sun guo ru ru ru ru sun sun sun ding uplcesiqtofmsms root tuber sun continued on next page ofethnopharmacology26420211132476 0ct ji table continued name ginsenoside rh2 vinaginsenoside r1 amino acid and derivatives phenylalanine pyroglutamic acid glutimic acid hexose tryptophan lglutamic acid detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part reference sun sun sun sun sun sun sun respectively a carboline by comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and scid0 trolline the chemical structures were shown in fig identified as indole anic acids and derivatives the biologically essential anic acids have been isolated and characterized from t hemsleyanum as well ten anic acids and seventeen fatty acids were identified from the aerial parts and root tuber of t hemsleyanum most of which were found in the aerial parts except stearic acid propanoic acid and dihydroxy octadecenoic acid all the anic acids and fatty acids are listed in no112“ and no95“ of table respectively terpenoids and steroids terpenoids and steroids are other kinds of secondary metabolites of t hemsleyanum thirteen of these compounds have been isolated and identified no122“ table liu yang liu isolated and identified αamyrine sitosterol ergosterol taraxerone taraxerol from the aerial part of t hemsleyanum in addition daucosterol campesterol stigmasterol 6obenzoyldaucosterol pteroside z ganoderic acid h 3epipapyriferic acid and oleanic acid were successively separated tuber roots of t hemsleyanum liu and yang from the inanic elements the mineral elements of tcm are indispensable supplements to the bioactive components which are closely related to the efficacy toxicity and side effects of tcm wu wu et al demonstrated that t hemsleyanum contained twentyseven different mineral elements namely li be na mg al k ca v cr mn fe co ni cu zn ga as se rb ag cd cs ba hg ti pb u moreover ca cu ni ba al k have higher loading values which are the characteristic elements of t hemsleyanum wang wang has indicated that the contents of fe mn zn and cu in three populations of t hemsleyanum cultivated in different environments were “ “ “ “ mg kgcid0 respectively pharmacology the ethnomedical uses of t hemsleyanum have stimulated various pharmacological studies on it the extracts and isolated compounds from t hemsleyanum showed a variety of bioactivities such as antiviral antibacterial antioxidant antipyretic analgesic hepatoprotective immunoregulatory and antitumor activity the detailed pharmacological activities of t hemsleyanum were presented in table and summarized as follows antiviral activity according to yang™s literatures yang the nitrogenous alkalicontaining extract a ketonecontaining extract f crude extract s1 and crude extract s2 of t hemsleyanum had different antiviral effect on mice and chicken embryo fibroblast cef infected with hemagglutinating virus of japan hvj influenza virus pr6 vesicular stomatitis virus vsv specifically s2 strongly inhibited the proliferation of influenza virus pr6 with at the concentration of mgml and mgml s1 has obvious antiviral effect on hvj at the concentrations of mgml and mgml both f and s1 displayed a strong suppressive effect on the plaque formation of vsv in vivo a f s1 s2 have different degrees of antiviral activity when the concentration of a was gkg the protective rate was up to and that of s1 gkg was however the author did not give the sample preparation method ding had demonstrated compounds quercetin3orutinoside kaempferol kaempferol3glucoside quercitrin quercetin kaempferol3orutinoside procyanidin dimmer and epicatechin which were isolated from t hemsleyanum were positively related to the activity of t hemsleyanum against h1n1 influenza virus the ethyl acetate extracts of t hemsleyanum have been shown to obviously restrain the secretion of hbsag and hbeag released by hbv with the ic50 values of “ mgl however the specific mechanism of action needs to be further confirmed yang and wu wang had proved that the nbutanol and ethyl acetate extraction of t hemsleyanum had antiviral activity against rsv and were superior to ribavirin with the ec50 values of mgl wang moreover the t hemsleyanum extracts had different degrees of inhibition to different hiv1 strains the ec50 values were between μgml and μgml and the
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introduction postoperative ileus poi a common complication after surgery severely affects postoperative recovery it is unclear whether pretreatment with transcutaneous electrical acupoint stimulation teas can improve recovery from poi this trial will evaluate the effects of pretreatment with teas on poimethods and analysis this will be a prospective randomised controlled trial american society of anesthesiologists asa physical status classification i“iii level patients aged “ years and scheduled for laparoscopic colon surgery will be included in the study it is planned that subjects will be randomised to the teas and sham teas steas groups the groups will undergo two sessions of teassteas daily for days before surgery with a final teassteas treatment min before anaesthesia the primary endpoint of the study will be time to first defaecation secondary endpoints will include time to first flatus time to tolerance of oral diet gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to independent walking length of hospital stay postoperative pain visual analogue scale score on the first days after surgery analgesic requirements complications and plasma concentrations of interferon ifn ifnÎ interleukin6 il6 and il1 multiple linear regression will be used to identify independent predictors of outcome measuresethics and dissemination this study has been approved by the chinese registered clinical trial ethics review committee no chiecrct20170084 the results of the trial will be published in an international peer reviewed trial registration number this study has been registered with the chinese clinical trial registry no chictr inr17013184trial status the study was in the recruitment phase at the time of manuscript submissionintroductionpostoperative dysfunction ileus poi of gastrointestinal is a transient gi strengths and limitations of this study –º this study aims to evaluate whether pretreatment with transcutaneous electrical acupoint stimulation teas can prevent postoperative ileus poi –º teas is a safe non invasive and easily accepted adjunctive intervention –º this study will provide deeper insights into the mechanism by which teas pretreatment reduces the inflammatory response –º this is a single centre study which is a potential limitationpropulsion that often occurs after abdominal surgery and may also occur after surgery at other sites1 the main symptoms of poi include abdominal pain and distention nausea vomiting difficult defaecation and intolerance to solid food poi is usually temporary but if prolonged may lead to surgical incision dehiscence intestinal anastomotic fistula abdominal cavity infection intestinal ischaemia aspiration pneumonia and other serious complications2“ a retrospective cohort study involving nearly hospitals in the usa showed that poi is a key reason for prolonged hospitalisation and increased medical costs for patients undergoing abdominal surgery1 the usa spends more than billion treating poi every year5 at present the most common methods used to treat poi include rational perioperative use of narcotic drugs and opioids eating as soon as possible after surgery avoidance of nasogastric tubes after the operation early ambulation postoperative epidural analgesia restriction of fluid intake the use wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access of minimally invasive surgery such as laparoscopic drug therapy and the use of chewing gum despite the numerous treatment strategies poi remains a difficult clinical challenge that compromises the rapid recovery of postoperative patients it is therefore necessary to find more effective convenient and economical treatment methods6“the main mechanism underlying poi may be activation of macrophages in the external muscular layer during the surgical procedure11 intestinal manipulation during surgery can activate macrophages in the outer muscle layer of the small intestine leading to release of inflammatory factors interleukin6 il6 il1 and the chemokine mip1α together with increased expression of the adhesion molecule icam1 on endothelial cells and induction of neutrophils and monocytes in the circulation into the small intestine muscle layer these cells and activated macrophages can release a large amount of inducible nitric oxide synthase and prostaglandin which inhibit the movement and contraction of the gi tract12 transport of these inflammatory mediators in the bloodstream causes activation of macrophages in the distal gi tract leading to poi over the entire intestinal tract14 it has been confirmed by a large number of animal experiments that reducing the inflammatory response is an effective way to treat poi15“there is a long history in traditional chinese medicine tcm of using acupuncture to treat functional gi diseases and in recent years there has been significant global interest in the beneficial effects of acupuncture on poi the positive effect of electroacupuncture ea on poi has been clearly demonstrated ng used ea to treat poi in patients undergoing laparoscopic colon surgery18 defaecation time and length of hospital stay were significantly shortened in patients who received ea compared with those who did not receive the treatment in patients undergoing hepatic resection you found a significant reduction in the incidence of poi in patients treated with a combination of acupuncture and chinese herbal medicine the length of hospitalisation was also significantly shortened in the treated group ± days vs ± days p001419in the previous studies we proved that pretreatment with acupuncture could reduce excessive activation of the innate immune system and inhibit the inflammatory response this effect may be achieved by activation of the vagal nervous system20 other studies have shown that transcutaneous electrical acupoint stimulation teas and ea have similar effects in the treatment of pain and alleviating the inflammatory response22 tcm holds that the best treatment for disease is prevention based on all of the above studies we hypothesise that the use of teas as a preoperative treatment may reduce the incidence of poi there have so far not been any studies that address this questionwe have therefore designed a randomised controlled trial to investigate whether pretreatment with teas can reduce the incidence of poi in patients undergoing laparoscopic colon resection the study is also designed to verify that the anti inflammatory effect is associated with the immunomodulatory function of teasmethods and analysisstudy objectivethe primary objective is to assess the effect of teas on clinical recovery of bowel function after laparoscopic colon surgery the secondary objective is to verify that suppression of overactivation of the innate immune system and reduction of the inflammatory response are the mechanisms underlying the ability of pretreatment of percutaneous acupuncture to prevent poistudy locationa prospective single centre double blinded randomised controlled trial will be conducted at shuguang hospital which is affiliated to the shanghai university of traditional chinese medicine chinastudy populationparticipants will be recruited according to the inclusion and exclusion criteriainclusion criteria male and female patients aged “ years patients undergoing elective laparoscopic colonic surgery and upper rectal resection such as left collect right colectomy and anterior resection of the upper part of the rectum and lower part of the sigmoid body mass index “ kgm2 asa classification i“iii patients provide signed informed consent the consent form can be viewed in online supplementary appendix exclusion criteria middle and lower rectal resection totalproctocolectomy or the need for complex endoscopic surgery need for abdominal wall fistula gi fistula fistula surgery or stoma creation history of abdominalpelvic operations or complications patients receiving epidural anaesthesia or epidural analgesia patients with skin infections surgical incision or scar at the point of application of acupuncture patients have a history of limb surgery spinal surgery or nerve injury patients who participated in other clinical trials or received other acupuncture therapy in the previous weeks patients with cardiac pacemakers patients have one of the following conditions before surgery chronic pain drug addiction or alcohol dependence patients with preoperative combination of severe central nervous system disease and severe mental illnesswang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cendpointsprimary endpointfirst defaecation time h that is time to first anal defaecation after laparoscopic surgerysecondary endpointstime to first flatus h time to tolerance of solid oral diet h gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to walk independently h length of hospital stay defined as the number of days from operation to discharge d criteria for hospital discharge include stability of vital signs with no fever achievement of flatus or defaecation ability to tolerate solid food without vomiting control of postoperative pain absence of other postoperative complications and ability to function at home independently or with home care provided pain will be assessed using the visual analogue scale vas on postoperative days and scale of to where represents complete absence of pain and represents the worst pain intensity postoperative requirements for analgesia will also be assessed inflammatory mediators interferon ifn ifnÎ interleukin6 il6 and il1 in blood will be measured before teassteas intervention and on days and after the operation postoperative complications will be recorded using the clavien dindo classification for complication assessment24 the follow up period will be at least monthswe add gi2 as a secondary outcome to the original protocol after recruitment of the study had already begun gi2 is a time indicator which will be calculated from two existing outcomes time to first defaecation and time to tolerance of oral diet there will be no harm to subjects no additional cost and no more workopen accessrandomisation and blindingpatients will be randomised to receive either teas or steas by stratified randomisation according to sex in a ratio figure using a computer generated random sequence a sealed envelope will be opened to determine to which group the patient has been assigned the acupuncturist will be aware of the treatment group patients as well as the outcome investigator nurse anaesthetist will be blinded to the treatment allocationcurrent sample size justificationaccording to wang jian and song jiangang™s preliminary study of teas pretreatment for prevention of poi in patients undergoing laparoscopic colon surgery in shuguang hospital the mean time to first defaecation following laparoscopic colon surgery was ± hours m±sd working on the assumption that a clinically meaningful difference in mean time to first defaecation between the teas and steas groups is day or hours patients would be needed in each group to reach a power of and a type i error rate if the dropout rate is a total sample size of patients for the two groups is needed for this studystatistical analysisdata for continuous variables ie first defaecation time first passage of flatus time to tolerance of oral diet time to walking independently length of hospital stay will be reported using the mean and sd m±sd for normally distributed data or median range for skewed data data for categorical variables will be expressed as a number percentage intergroup differences will be assessed using the student™s t test or mann whitney u test intergroup differences in inflammatory mediators at time points of pre teassteas treatment and on figure flowchart of the study protocolwang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access figure acupoints selected in this trial a hegu il4 and neiguan p6 b zusanli st36 and shangjuxu st37 c han™s acupoint nerve stimulatorpostoperative days and were assessed by two way repeated measures analysis of variance with bonferroni post hoc test the significance level will be set at all data will be analysed using spss v170 software or other appropriate statistical software packagespretreatmentpatients randomised to the teas and steas groups will undergo two treatment sessions daily for three consecutive days before surgery the patients will then be treated for a final time min before anaesthesiafor patients in the teas group the zusanli st36 shangjuxu st37 hegu li4 and neiguan p6 acupoints will be identified before electrical stimulation with surface electrodes figure selection of these acupoints is based on a consensus between the acupuncturists carrying out the study the acupuncturist will stimulate these acupoints using a han™s acupoint nerve stimulator hans200a nanjing jisheng medical technology nanjing china at a frequency of hz the intensity will be adjusted for each individual to maintain a slight twitching of the regional muscle and achieve de qi sensations such as soreness numbness distention and heaviness the steas group will receive a strong but comfortable current for s and the current will then gradually vanish over the next s25 the participants of both groups will be told that they are receiving current stimulation each session of acupoints treatment will last for min during the application of teas patients will be required not to change the current settings themselves a prompt beep at the end of teas will indicate the end of treatmentall surgery will be carried out under general anaesthesia using standardised anaesthetic procedures patients will be fasted for hours before surgery right upper extremity venous access will be established before the patients entering the operating theatre ringer™s lactate solution mlkg will be administered by intravenous infusion for compensatory expansion before induction of anaesthesia patients will then receive midazolam mgkg fentanyl µgkg vecuronium bromide mgkg and propofol “ mgkg intravenously for induction of anaesthesia anaesthesia will be maintained using a cp600 anaesthesia delivery system slgo medical technology beijing china the dose wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cof propofol will be adjusted to maintain the bispectral index in the range of “ after surgery all patients will remain in the post anaesthesia care unit and then return to the ward for recovery until dischargethe perioperative management of all patients will be standardised early ambulation will be encouraged and oral feeding will be resumed as early as possible all patients will be followed up for at least months after discharge from the hospitaladverse eventsall adverse reactions will be closely monitored through spontaneous reports by patients or direct observation by clinicians or by asking the patients about adverse events using open questions all adverse reactions will be recorded and appropriate treatment will be provided if necessary serious adverse events will be reported to the ethics committeedata collection and managementdemographic variables and clinical data will be collected from all patients during the study blood pressure heart rate and oxygen saturation will also be monitored any adverse events will be recorded data will be collected throughout the study and will be securely managed under conditions of confidentiality data collection will be performed by a nurse anaesthetist the participants will be referred to by their participant number rather than by their name throughout the study unless otherwise specified all relevant documents and files will be archived for years the data will be accessible only by investigators who sign the confidential disclosure agreement and by institutional or governmental auditors during the study data without patient identifiers will be publicly accessible after the study data collection and management will be monitored by the institutional ethics committee for clinical research of shuguang hospitalpatient and public involvementthis study is currently in the recruitment phase patients andor the public were not involved in study design or conduct of the study the participants will be able to access the study results through social mediadiscussionpoi continues to represent an important cause of morbidity after colon surgery the prevention of poi is thus of great importance in reducing perioperative complications and reducing hospitalisation costs although it has been shown that ea can shorten the duration of poi18 the effectiveness of teas which is a similar technique in preventing poi has not been investigated it is therefore important to assess the effectiveness of teas in preventing poi through a clinical studythis study has several strengths first the intervention strategy of the protocol will be pretreatment with teas previous studies have shown that pretreatment open accesshas a prophylactic effect for example pretreatment with teas has been shown to improve pain treatment26 and to improve resuscitation after anaesthesia with reduction of postoperative nausea and vomiting28 it is however unclear whether preoperative teas can prevent poi studies suggest that early preoperative intervention may be more beneficial in regulating physiological functions and preventing poi29 in an extension to these findings the present study will help to determine whether teas pretreatment could improvement poisecond the effectiveness of teas will be evaluated by assessing clinical function and by serological examination in this randomised controlled trial of patients undergoing laparoscopic colorectal surgery our aim is to assess the effects of preoperative teas on poi using relevant clinical parameters associated with bowel function these include time to first defaecation time to first flatus time to tolerance of oral diet and gi2 importantly we will also measure serum concentrations of inflammatory mediators associated with poi such as ifn ifnÎ il6 and il1 our findings may thus provide deeper insights into the mechanisms by which teas improves poithere are also limitations to this protocol various clinical indicators have been used in studies for the diagnosis of poi but there is no consensus on which clinical parameter is the best for assessment of gi transit9 two indicators that are widely used to assess bowel movement will be used in this study time to first defaecation will be the primary outcome and time to first flatus will be one of the secondary outcomes there is a possibility that we may observe conflicting results ie significant improvement in time to flatus but not defaecation because flatus can vary considerably between patients clinical trials support the time to tolerance of oral diet and gi2 defined as the later of the following two events time to first tolerance of solid food and time to first bowel movement as supplementary secondary outcomes to measure the recovery time of gi function and these will be used in this study32 other limitations of these indicators are that they require objective measurement of motility and are time consuming to measure34 recently this situation has been improved by the use of in vivo monitoring techniques to assess the function of gi movements innovative devices such as sitz markers have been used to evaluate postoperative recovery of small bowel movement by counting the number of sitz markers that did not pass through the ileocecal valve but remained in the small intestine using radiography36 the smartpill is a swallowable device that record parameters within the gi tract indicators such as ph temperature and intracavitary pressure can be collected to analyse gi transit times in vivo37 these devices acquire objective parameters to evaluate bowel movement and could save time research into the satisfaction of both doctors and patients with these device needs to be carried out furthermore this study is a single centre trial and because the therapeutic effect of teas may be affected by ethnicity and region it will wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access be necessary to conduct multicentre and large sample studies in the futurenotwithstanding its limitations this study can clearly indicate the overall effects of teas on postoperative recovery we hypothesise that pretreatment with teas could improve recovery of gi function in patients undergoing laparoscopic surgery if this study provides positive results it will be possible to recommend this pretreatment strategy for patients undergoing abdominal surgery relevant cost effectiveness studies are also worthy of considerationauthor affiliations1anesthesiology shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai china2anesthesiology wenzhou medical university the sixth affiliated hospital lishui china3research institute of acupuncture anesthesia shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai chinaacknowledgements we thank dr stanley tao from shanghai ruihui biotech for his valuable assistance in the statistical design of this studycontributors jw conceived the study dl wt jg and gf participated in its design and coordination wc yy ws and jg collected references and developed the protocol gy and ly will perform statistical analyses rf will follow up with patients and record data jw lf and js drafted the manuscript all authors have read and approved the final manuscriptfunding the present study is supported by the project of the national natural science foundation of china nos and and the commercial sponsorship of sinch pharmaceuticals techcompeting interests none declaredpatient consent for publication obtainedprovenance and peer review not commissioned externally peer reviewedopen access this is an open access article 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 idlihua a0fan http orcid org references iyer s saunders wb stemkowski s economic burden of postoperative ileus associated with colectomy in the united states j manag care pharm “ boelens pg heesakkers ffbm luyer mdp et a0al reduction of postoperative ileus by early enteral nutrition in patients undergoing major rectal surgery prospective randomized controlled trial ann surg “ melis m fichera a ferguson mk bowel necrosis associated with early jejunal tube feeding a complication of postoperative enteral nutrition arch surg “ moghadamyeghaneh z hwang gs hanna mh et a0al risk factors for prolonged ileus following colon surgery surg endosc “ goldstein jl matuszewski ka delaney cp et a0al inpatient economic burden of postoperative ileus associated with abdominal surgery in the united states p and t “ bragg d el sharkawy am psaltis e et a0al postoperative ileus recent developments in pathophysiology and management clin nutr “ wolthuis am bislenghi g fieuws s et a0al incidence of prolonged postoperative ileus after colorectal surgery a systematic review and meta analysis colorectal dis 201618o1“ nguyen dl maithel s nguyen et et a0al does alvimopan enhance return of bowel function in laparoscopic gastrointestinal surgery a meta analysis ann gastroenterol “studies and clinical aspects of gadolinium salts and chelates cardiovasc drug rev “ koscielny a kalff jc t helper cell type memory cells and postoperative ileus in the entire gut curr opin gastroenterol “ mikkelsen hb thuneberg l opop mice defective in production of functional colony stimulating factor1 lack macrophages in muscularis externa of the small intestine cell tissue res “ van bree shw nemethova a cailotto c et a0al new therapeutic strategies for postoperative ileus nat rev gastroenterol hepatol “ hilton wm lotan y parekh dj et a0al alvimopan for prevention of postoperative paralytic ileus in radical cystectomy patients a cost effectiveness analysis bju int “ wehner s behrendt ff lyutenski bn et a0al inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents gut “ wehner s straesser s vilz to et a0al inhibition of p38 mitogen activated protein kinase pathway as prophylaxis of postoperative ileus in mice gastroenterology “ schwarz nt kalff jc türler a et a0al prostanoid production via cox2 as a causative mechanism of rodent postoperative ileus gastroenterology “ engel dr koscielny a wehner s et a0al t helper type memory cells disseminate postoperative ileus over the entire intestinal tract nat med “ adding lc bannenberg gl gustafsson le basic experimental ng ssm leung ww mak twc et a0al electroacupuncture reduces duration of postoperative ileus after laparoscopic surgery for colorectal cancer gastroenterology “ you x m mo x s ma l et a0al randomized clinical trial comparing efficacy of simo decoction and acupuncture or chewing gum alone on postoperative ileus in patients with hepatocellular carcinoma after hepatectomy medicine 201594e1968 song jg li hh cao yf et a0al electroacupuncture improves survival in rats with lethal endotoxemia via the autonomic nervous system anesthesiology “ zhang j yong y li x et a0al vagal modulation of high mobility group box1 protein mediates electroacupuncture induced cardioprotection in ischemia reperfusion injury sci rep balogun ja biasci s han l the effects of acupuncture electroneedling and transcutaneous electrical stimulation therapies on peripheral haemodynamic functioning disabil rehabil “ jiang y wang h liu z et a0al manipulation of and sustained effects on the human brain induced by different modalities of acupuncture an fmri study plos one 20138e66815 dindo d demartines n clavien p a classification of surgical complications a new proposal with evaluation in a cohort of patients and results of a survey ann surg “ rakel b cooper n adams hj et a0al a new transient sham tens device allows for investigator blinding while delivering a true placebo treatment j pain “ huang l pan y chen s et a0al prevention of propofol injection related pain using pretreatment transcutaneous electrical acupoint stimulation turk j med sci “ zhang q gao z wang h et a0al the effect of pre treatment with transcutaneous electrical acupoint stimulation on the quality of recovery after ambulatory breast surgery a prospective randomised controlled trial anaesthesia “ zheng lh sun h wang gn et a0al effect of transcutaneous electrical acupoint stimulation on nausea and vomiting induced by patient controlled intravenous analgesia with tramadol chin j integr med “ stakenborg n labeeuw e gomez pinilla pj et a0al preoperative administration of the ht4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons gut “ vather r trivedi s bissett i defining postoperative ileus results of a systematic review and global survey j gastrointest surg “ wu z boersema gsa dereci a et a0al clinical endpoint early detection and differential diagnosis of postoperative ileus a systematic review of the literature eur surg res “ deng g wong wd guillem j et a0al a phase ii randomized controlled trial of acupuncture for reduction of postcolectomy ileus ann surg oncol “ van bree shw bemelman wa hollmann mw et a0al identification of clinical outcome measures for recovery of gastrointestinal motility in postoperative ileus ann surg “wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c maffezzini m campodonico f canepa g et a0al current perioperative management of radical cystectomy with intestinal urinary reconstruction for muscle invasive bladder cancer and reduction of the incidence of postoperative ileus surg oncol “ bungard tj kale pradhan pb prokinetic agents for the treatment of postoperative ileus in adults a review of the literature pharmacotherapy “open access chae h d kwak m a kim i h effect of acupuncture on reducing duration of postoperative ileus after gastrectomy in patients with gastric cancer a pilot study using sitz marker j altern complement med “ vilz to pantelis d lingohr p et a0al smartpill® as an objective parameter for determination of severity and duration of postoperative ileus study protocol of a prospective two arm open label trial the pidusa study bmj open 20166e011014wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c'
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purposeconjunctival squamous cell carcinoma scc is primarily treated with surgical resectionscc has various stages and local recurrence is common the purpose of this study was todetermine molecular localization of epidermal growth factor receptor egfr and the possibility of egfr as a biomarker for the management of conjunctival sccmethodsin this retrospective study we performed immunohistochemistry to evaluate egfr expression and localization in tumor cells egfr mutationspecific expression e746a750del andl858r and human papillomavirus expression in a series of conjunctival sccsresultsall tumors in our cohort were egfr positive twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining in additionwe calculated the percentages of the two most important mutations in egfr exon a750del exon l858r mutant in conjunctival sccs weobserved that the translocation of egfr from the membrane into the cytoplasm was relatedto clinical prognosis as we detected correlations between egfr cytoplasmic staining andfinal orbital exenteration and between decreased egfr membrane staining and progressionfree survivala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation sakai a tagami m kakehashi akatsuyamayoshikawa a misawa n wanibuchi h expression intracellular localizationand mutation of egfr in conjunctival squamouscell carcinoma and the association with prognosisand treatment one e0238120 101371 pone0238120editor sanjoy bhattacharya bascom palmer eyeinstitute united statesreceived april accepted august 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 pone0238120copyright sakai 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 supporting informationfiles one 101371 pone0238120 august one 0cfunding none of the authors have any proprietaryor financial interests to declarecompeting interests none of the authors haveany proprietary or financial interests to declaresegfr is important in the pathology of ocular surface squamous neoplasia including sccand is a prognostic factor increased understanding of egfr mutations may have importantimplications for future treatment optionsegfr in conjunctival squamous cell carcinomaintroductionocular surface squamous neoplasia ossn includes several diseases such as conjunctival premalignant dysplasia carcinoma in situ and invasive conjunctival squamous cell carcinomascc the annual incidence of ossn was casesmillionyear conjunctival intraepithelial neoplasia casesmillionyear scc casesmillionyear in the united kingdom [ ] in the united states the rate of scc is 5fold higher among males and whites other previous research revealed that the risk increases with exposure to direct daylightand in outdoor workers metaanalysis demonstrated an association with human immunodeficiency virus odds ratio and human papillomavirus hpv odds ratio howeverno large epidemiological studies have been performed on people living in the far eastscholz examined clinicopathological factors and biomarkers and identified promotermutations in telomerase reverse transcriptase in of samples of conjunctival ossn associated with ultraviolet light induction recent research demonstrated that pdl1 isexpressed in almost half of conjunctival scc cases and noted the potential application ofimmune checkpoint blockade as a treatment strategy for conjunctival scc molecular targeted therapy is now used to treat most carcinomas and its use is continuingto increase uveal melanoma also has recently been reported in the ocular oncology area gefitinib is a relatively old tyrosine kinase inhibiter tki that is used as a molecular targeted therapy and its effects have been reported in various carcinomas on the other hand nobasic clinical studies on ocular tumors have been reported [“] in our current study weinvestigated epidermal growth factor receptor egfr expression in our cases to assess thepossible effect of gefitinib we also examined the molecular expression and intracellular localization of egfr in conjunctival scc in east asian patientsmaterials and methodsselection of cases and collation of clinicopathologic datathis study was approved by the institutional review boards of osaka city university andkobe kaisei hospital and adhered to the tenets of the declaration of helsinki writteninformed consent was obtained from all patients before enrollment we identified patientstreated by ophthalmologists aa mt between november and july from whom wewere able to procure tissue blocks with residual tumor for each patient we collected demographic features age at initial diagnosis and at presentation to our institution and sex andprimary tumor features disease status at presentation [primary or recurrent] and in situ versusinvasive disease the american joint committee on cancer ajcc stage local recurrenceanatomic site and date metastases regional or distant and date vital status at last followup cause of death types of surgery and adjuvant therapy were also recordedimmunohistochemistry ihcimmunohistochemical studies for egfr and hpv were performed on 6μmthick tissue sections using the following antibodies antihuman egfr rabbit monoclonal antibody clone one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomasp84 414r14 cell marque rocklin ca usa antihpv mouse monoclonal antibodyclone k1h8 ab75574 abcam cambridge uk horseradish peroxidaseconjugated antirabbit igg hl goat polyclonal antibody histofine nichirei corporation tokyojapan and horseradish peroxidaseconjugated antimouse igg hl goat polyclonal antibody histofine nichirei corporationegfr mutationspecific immunohistochemical staining was performed on cases as primary antibodies we used egfr e746a750del cell signaling technologies danversma usa and egfr l858r cell signaling technologies which were manuallyapplied to the slides stained sections were viewed with an olympus bx53dp74as controls for staining benign conjunctival lesions were also stained for egfr and coloncancer samples were stained as a positive controlimage analysisslides immunostained for egfr egfr mutations and hpv were evaluated in a blinded manner by two specialists mt and ak egfr expression was visually estimated as the percentageof tumor cells with complete or partial membranous staining tumors with egfr staining in� of tumor cells were considered the diffuse staining type diffuse type and those with of tumor cells were considered the focal staining type focal type the presence orabsence and intensity of cell membrane staining were semiquantitatively divided into groupswith a score of “ none weak strong very strong the presence or absence andintensity of cell cytoplasmic staining were also divided into groups with a score of “ andsemiquantitatively analyzed none weak strong very strong egfr mutationspecific immunostaining was divided into two groups those with immunostaining that wasclearly present and those without immunostainingslides immunostained for hpv were assessed with visual evaluation for the presence ofpunctate nuclear signals within tumor nuclei at — magnification and were scored as positive or negativeegfr expression in tumorsegfr expression in the tumor was analyzed with nanostring analysis archival formalinfixedparaffinembedded tumor tissue was retrieved and manually macrodissected total mrnawas isolated from the macrodissected tumor tissues using a qiagen mirneasy kit qiagenvalencia ca usa according to the manufacturer™s instructions the rna sample was quantified with nanodrop thermo scientific wilmington de usa and regarded as adequate ifit contained ng at minimum the sample was subsequently analyzed with the ncounterpancancer progression panel nanostring seattle wa usa according to the manufacturer™s instructions nanostring data processing was done with the r statistical programmingenvironment v342 considering the counts obtained for positive control probe sets rawnanostring counts for each gene were subjected to technical factorial normalization whichwas carried out by subtracting the mean counts plus two times the standard deviation from thecodeset inherent negative controls subsequently biological normalization using the includedmrna reference genes was performed additionally all counts with p after a onesidedttest versus negative controls plus two times the standard deviation were interpreted as notexpressed over basal noisestatistical analysisthe clinical and histopathologic characteristics were summarized using descriptive statisticscorrelations between immunohistochemical demographic and clinicopathologic factors were one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomaassessed using the wilcoxon rank sum and fisher™s exact tests progressionfree survival pfswas defined as the time from surgery to disease recurrence or death from any cause coxregression modeling was used to evaluate correlations between clinicopathologic and immunohistochemical features and survival outcomes statistical analyses were performed usingspss statistics version software ibm japan tokyo japan values of p were considered statistically significantresultsclinicopathologic findings of our cohort are summarized in table all patients in ourcohort were east asian and included men and women with a mean age at presentation of years fourteen patients had invasive scc and had an in situtumor primary orbital exenteration was necessary for local disease control in two patients and two patients underwent additional orbital exenteration nine patients table clinicopathologic findings of cases of conjunctival squamous cell carcinomaage years mean rangesexmalefemalefollowup after primary surgery months rangetstage ajccall n n “ “tist1t2t3t4primary surgery typelocal excisionorbital exenterationadjuvant therapynoyesadditional excisiontopical chemotherapyradiation therapyimmunohistochemical markershpv status in tumor cellsnegativepositiveegfr expression in tumorsdiffuse stainingfocal stainingnegativecell membrane egfr expression in tumorsvery strongstrongweak one 101371 pone0238120 august continued one 0ctable continuednegativecell cytoplasm egfr expression in tumorsvery strongstrongweaknegativeoutcomeorbital exenterationyesnolocal recurrence after curative therapyyesnometastasisdistantregional distantregionalnonevital status at last followupdeadalivecause of deathconjunctival scc metastasisother101371 pone0238120t001egfr in conjunctival squamous cell carcinomaall n n underwent adjuvant therapy most commonly additional local surgery topical chemotherapyand radiation therapy were performed in one patient in the adjuvant therapy group of thisgroup one patient died with disease months after diagnosis of regional and lung metastasesthe other patient was alive without disease at months after diagnosis of regional metastasestwo patients died one of which was due to conjunctival scc described above ninepatients experienced local recurrence after curative surgeryall tumors were egfr positive in our cohort twentyone of tumors showed focal egfr staining and seven showed diffuse egfr staining fig analysisof egfr intracellular staining patterns showed scores of for membrane staining and for cytoplasmic staining no significant difference was found between carcinoma in situ tisand invasive carcinoma tadv table no significant difference was found in the scoredepending on the stage egfr expression in colon cancer was used as a positive control fig2aon the other hand seven benign conjunctival lesions three pinguecula three pterygiumone dermoid cyst showed partial weak positive staining in conjunctival squamous epithelialcells especially on the cell membrane fig 2b in addition cytoplasmic staining was seen inonly one case benign cases showed scores of for membrane staining and for cytoplasmic staining cytoplasmic staining patterns were significantly different in benign compared to scc cases p table the correlation between egfr staining focal ordiffuse and egfr localization cytoplasmic staining group was not significantly different one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr expression in conjunctival scc focal egfr staining a and diffuse egfr staining b scale bar μm inset corresponding field in ahematoxylineosinstained section membrane staining very strong c and cytoplasm staining very strong d scale bar μm101371 pone0238120g001but the diffuse egfr group tended to have a higher score p and respectivelytable egfr e746a750 del and egfr l858r expression were assessed with immunohistochemistry in all patients fig the mutation at exon egfr e7446a750 del was confirmedin cases and that at exon egfr l858r point mutation was confirmed in cases with ihc table the relationship between egfr mutation and egfr stainingtable staining patterns of egfrcell membranetis in situtadv invasiven n benign tumorn cell cytoplasmtis in situtadv invasiven n benign tumorn �p value based on the nonpaired ttest101371 pone0238120t002staining patterns n totaltotalaverageaveragepp� one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig a egfr expression in colon cancer as a positive control scale bar μm b egfr expression in a control benign lesion pinguecula scale bar μminset corresponding field in a hematoxylineosinstained section101371 pone0238120g002focal or diffuse was determined using univariate linear regression analysis with correctionfor age p regarding egfr expression in tumors we compared the tis and tadv groups according toajcc t grading n4 no significant difference was found p fig the majority of patients in our cohort were hpv negative n table the positive rate of hpv immunoreactivity increased with increases in ajcc t grading but the correlation was not statistically significantthe cox regression model was used to examine and analyze the relationship between longterm prognosis including orbital exenteration and pfs and the clinicopathological statusegfr staining pattern and egfr mutation univariate cox regression analyses revealed significant correlations between egfr cytoplasmic staining and final orbital exenteration hazard ratio hr p table additionally a significant correlation was seenbetween the t stage ajcc and pfs and between egfr membrane staining and pfs hr p p respectively table local recurrence distant metastasisrate and overall survival rate were not statistically significant in addition the egfr mutationwas not significantly correlated with final orbital exenteration or pfs tables and discussionto the best of our knowledge this is one of the first studies to survey the prevalence of egfrmutations and intracellular localization in conjunctival scc and to evaluate the prognostic significance of tumor cells that express egfr in conjunctival sccin this study we found that the tumor tissue of all conjunctival sccs expressedegfr in addition we determined the percentages of the two most important mutations intable egfr staining and localizationcell membranecell cytoplasmicegfr focaln ±±egfr diffusen ±±p101371 pone0238120t003 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig egfr mutationspecific expression in conjunctival scc a basement membrane staining in a tumor with egfr e746a750 del bwhole tumor staining in an egfr e746a750 del mutant c conjunctival scc layer cells with strong staining in an egfr l858r mutant scalebar μm101371 pone0238120g003egfr exon 746a750del exon l858r mutant in conjunctival sccs we also showed that the translocation of egfr from the membrane into the cytoplasm was related to clinical activation of cancer as correlations between egfr cytoplasmicstaining and final orbital exenteration and between decreased egfr membrane staining andpfs were noted although the number of cases examined was small the expression of cytoplasmic staining of egfr was weak but significantly different from membrane staining in thebenign disease group our hypothesis is that as egfr transitions from the membrane into thecytoplasm malignant changes progress in addition a correlation between egfr stainingfocal or diffuse and egfr cytoplasmic staining was seen and a higher score tended to bepresent in the diffuse egfr staining grouptable summary of egfr e746a750 del and egfr l858r point mutationsmutationn age y sex mt stage egfr staining patterns diffuseegfr localization score membraneexon egfr e746a750 del n fexon egfr l858r point mutationn t3 t2 tis t3 tis focalcytoplasmicm male f female101371 pone0238120t004 one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig for egfr expression in tumors we compared carcinoma in situ tis and invasive carcinoma tadv groups according toajcc t grading n4 ns not significant101371 pone0238120g004intracellular transfer of egfr in the group with diffuse staining may indicate progressionand although no statistical differences were observed in this study significant findings mayemerge by increasing the number of cases in the futurein the past especially in african countries several studies on conjunctival sccs and egfrexpression have been reported they suggested a potential association with hpv [ ]other previous studies reported that posttranslational modification can promote egfrtable relationship between orbital exenteration and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750 del 8exon l858r point mutation positive 7negative hr — ci““““ — ““““ — p�ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t005 one 101371 pone0238120 august one 0ctable relationship between pfs and clinicopathologic and molecular factorsunivariate analysisvariablesagesextstage ajccegfr stainingn mean yearsmale 15female tis t1 t2 21t3 �focal 21diffuse egfr membrane stainingvery strong 1strong 21weak 7negative egfr cytoplasmic stainingegfr mutationhpv positivevery strong 4strong 6weak 19negative exon e746a750del 8exon l858r point mutation positive 7negative ci indicates confidence interval hr hazard ratestatistically significant differences are underlined�p value based on the cox proportional hazard model101371 pone0238120t006egfr in conjunctival squamous cell carcinomahr ci“““ — ““““ — “p��endocytosis and lysosomal degradation of egfr thereby ensuring termination of receptor signaling [ ]in our cohort expression and localization of egfr and its association with prognosis werefirst reported in the asian race additionally intracellular translocation of egfr from membrane staining to cytoplasm staining likely by endocytosis was associated with the percent offinal orbital exenteration cytoplasmic staining hr p and pfs membranestaining hr p in our cohort regarding the difference in local changes inegfr immunoreactivity in patients without egfr expression in the tumor we compared thetis and tadv groups according to ajcc t grading a recent study showed that feedback regulatory loops can modulate growth factors and receptor tyrosine kinases such as egfr to regulate cellular functions including abnormal states such as cancer our study examined thisphenomenon clinically and confirmed a pathological difference without changes in geneexpressionegfr mutations in ossn including invasive sccs have not been examined in asianpatients since approximately egfr mutations in lung cancer had been registeredin the cosmic the catalog of somatic mutations in cancer database most are concentrated in the exon “ region of the intracellular tyrosine kinase domain the most frequentone is at codon of exon a deletion mutation is present at a site centered on five aminoacids elrea near amino acid and a point mutation changes leucine to argininel858r at codon of exon shigematsu in and mitsudomi in reported that egfr mutations are common in asians females nonsmokers and adenocarcinomas in lung cancer [ ] generally when egfr mutation occurs the tyrosine kinaseactivity of egfr at the atp binding site is constantly active even without growth factor cancer cell growth and survival depend on this pathway oncogene addiction egfr tkis competitively inhibit atp binding in the kinase domain and suppress autophosphorylation ofegfr blockade of signal transmission has antitumor effects previous reports of egfractivating mutations common mutations described the frequency of exon deletion mutations as and for l858r mutations in lung cancer [ ]egfr mutations were examined to verify the effect of gefitinib on positive nonsmall celllung carcinoma in two phase iii clinical trials from japan in the nej002 trial and thewjtog3405 trial gefitinib was the test treatment group the standard treatment in the former one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomafig schematic of movement of egfr into the cytoplasm by endocytosis to avoid excessive signaling and for recycling101371 pone0238120g005was carboplatin paclitaxel and in the latter was cisplatin in all studies the gefitinib groupshowed superior pfs [ ] in view of these findings in lung cancer in asians our findingsregarding egfr expression and mutations will provide further options for potential treatmentof ossn for pre and postsurgical treatmentthe association of scc with hpv was not confirmed because the number of cases wassmall in addition our results may not be accurate because we did not use multiplex pcrwhich is currently the most suitable genotyping method ours is the first report to show that differences in the expression form and mutations inegfr in ossn are associated with prognosis and treatmentin an animal model egfr inhibition affected epithelial cell proliferation and stratificationduring corneal epithelial wound healing and may play a role in maintaining normal cornealepithelial thickness gefitinib is an egfr inhibitor and is the first approved molecular targeted therapy for cancer treatment in japan thus understanding the pathological role of egfr in ossn andapplying it to treatment are of great significance for seeking new treatment indications inossn including conjunctival sccs in this study egfr may translocate from the cell membrane into the cytoplasm tumor cells may transfer egfr into the cytoplasm by endocytosisto avoid excessive signaling by the feedback system fig furthermore in this study theegfr mutation was present in many patients with ossn this finding may suggest a courseof treatment in the future in addition the method we used for identification of egfr mutations was not general genotyping but was a judgment of immunohistochemically stained sections although the sensitivity and specificity were high in a previous report this is still alimitation one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinomathis study has important limitations first regarding egfr expression on the ocular surface changes in benign diseases and agerelated changes in normal tissues may not have beensufficiently investigated our study found that egfr mutations were also present in conjunctival scc in east asians however we did not obtain results that correlated with the final prognosis further studies including further multiinstitutional studies and an increase in thenumber of cases will be needed in the future another limitation is that double testing of formalinfixed paraffinembedded specimens and plasma with realtime pcr for detection ofegfr mutations is more common than ihc in actual clinical practice according to the literature both the sensitivity and specificity were satisfactory for these two types of mutations in addition the size of our study cohort was small n and the length of followup lessthan year in some patients may not have been sufficient for longterm outcome analysestherefore additional studies will be needed to corroborate our findingsin the results of this study indicate that egfr is an active molecular target inthe pathology of ossn including scc and is a prognostic factor the finding also suggests thatdiscovery of mutations may have important implications for future treatment optionssupporting informations1 filexlsxacknowledgmentswe gratefully acknowledge the technical assistance of the research support platform osakacity university graduate school of medicine and the clinical laboratory department of kobekaisei hospitalauthor contributionsconceptualization mizuki tagami atsushi azumidata curation atsushi sakai mizuki tagami atsuko katsuyamayoshikawa norihiko misawa atsushi azumiformal analysis mizuki tagami anna kakehashi norihiko misawafunding acquisition mizuki tagamiinvestigation mizuki tagami atsuko katsuyamayoshikawa atsushi azumimethodology mizuki tagami anna kakehashi atsuko katsuyamayoshikawa atsushiazumiproject administration mizuki tagamisupervision anna kakehashi hideki wanibuchi atsushi azumi shigeru hondavisualization atsushi sakai mizuki tagami anna kakehashiwriting “ original draft atsushi sakai mizuki tagamiwriting “ review editing mizuki tagami shigeru hondareferenceslee ga hirst lw ocular surface squamous neoplasia surv ophthalmol “ 101016s0039625705800542 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kiire ca stewart rmk srinivasan s heimann h kaye sb dhillon b a prospective study of the incidence associations and outcomes of ocular surface squamous neoplasia in the united kingdom eyelond “ mcclellan aj mcclellan al pezon cf karp cl feuer w galor a epidemiology of ocular surfacesquamous neoplasia in a veterans affairs population cornea “ 101097ico0b013e31829e3c80 pmid sun ec fears tr goedert jj epidemiology of squamous cell conjunctival cancer cancer epidemiolbiomarkers prev “ pmid scholz sl thomasen h reis h frequent tert promoter mutations in ocular surface squamousneoplasia invest ophthalmol vis sci “ 101167iovs1517469pmid nagarajan p elhadad c gruschkus sk ning j hudgens cw sagiv o pdl1pd1 expressioncomposition of tumorassociated immune infiltrate and hpv status in conjunctival squamous cellcarcinoma invest ophthalmol vis sci “ 101167iovs1926894pmid le tourneau c delord jp gonc¸alves a gavoille c dubot c isambert n molecularly targetedtherapy based on tumour molecular profiling versus conventional therapy for advanced cancershiva a multicentre openlabel proofofconcept randomised controlled phase trial lancetoncol “ 101016s1470204515001886 pmid el zaoui i bucher m rimoldi d nicolas m kaya g pescini gobert r conjunctival melanomatargeted therapy mapk and pi3kmtor pathways inhibition invest ophthalmol vis sci “ 101167iovs1826508 pmid ciardiello f tortora g a novel approach in the treatment of cancer targeting the epidermal growth factor receptor clin cancer res “ pmid lynch tj bell dw sordella r gurubhagavatula s okimoto ra brannigan bw activating mutations in the epidermal growth factor receptor underlying responsiveness of nonsmallcell lung cancer togefitinib n engl j med “ 101056nejmoa040938 pmid paez jg ja¨nne pa lee jc tracy s greulich h gabriel s egfr mutations in lung cancer correlation with clinical response to gefitinib therapy science “ 101126science1099314 pmid cesano a ncounter® pancancer immune profiling panel nanostring technologies inc seattlewa j immunother cancer 101186s4042501500887 pmid yu jj fu p pink jj dawson d wasman j orem j hpv infection and egfr activationalteration in hivinfected east african patients with conjunctival carcinoma one e10477101371 pone0010477 pmid mwololo a nyagol j rogena e ochuk w kimani m onyango n correlation of egfr pegfrand p16ink4 expressions and high risk hpv infection in hivaidsrelated squamous cell carcinoma ofconjunctiva infect agent cancer 1011861750937897 pmid haglund k dikic i the role of ubiquitylation in receptor endocytosis and endosomal sorting j cell sci “ 101242jcs091280 pmid zhang x gureasko j shen k cole pa kuriyan j an allosteric mechanism for activation of the kinasedomain of epidermal growth factor receptor cell “ 101016jcell pmid avraham r yarden y feedback regulation of egfr signalling decision making by early and delayedloops nat rev mol cell biol “ 101038nrm3048 pmid kobayashi y mitsudomi t not all epidermal growth factor receptor mutations in lung cancer are created equal perspectives for individualized treatment strategy cancer sci “101111cas12996 pmid shigematsu h lin l takahashi t nomura m suzuki m wistuba ii clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers j natl cancerinst “ 101093jncidji055 pmid mitsudomi t yatabe y mutations of the epidermal growth factor receptor gene and related genes asdeterminants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancercancer sci “ 101111j13497006200700607x pmid yun ch mengwasser ke toms av woo ms greulich h wong kk the t790m mutation inegfr kinase causes drug resistance by increasing the affinity for atp proc natl acad sci u s a “ 101073pnas0709662105 pmid one 101371 pone0238120 august one 0cegfr in conjunctival squamous cell carcinoma kobayashi y togashi y yatabe y mizuuchi h jangchul p kondo c egfr exon mutationsin lung cancer molecular predictors of augmented sensitivity to afatinib or neratinib as comparedwith first or thirdgeneration tkis clin cancer res “ 10115810780432ccr151046 pmid wu jy yu cj chang yc yang ch shih jy yang pc effectiveness of tyrosine kinase inhibitors onuncommon epidermal growth factor receptor mutations of unknown clinical significance in nonsmallcell lung cancer clin cancer res “ 10115810780432ccr10 pmid maemondo m inoue a kobayashi k sugawara s oizumi s isobe h gefitinib or chemotherapyfor nonsmallcell lung cancer with mutated egfr n engl j med “ 101056nejmoa0909530 pmid mitsudomi t morita s yatabe y negoro s okamoto i tsurutani j gefitinib versus cisplatin plusdocetaxel in patients with nonsmallcell lung cancer harbouring mutations of the epidermal growth factor receptor wjtog3405 an open label randomised phase trial lancet oncol “101016s147020450970364x pmid nishiwaki m yamamoto t tone s murai t ohkawara t matsunami t genotyping of humanpapillomaviruses by a novel onestep typing method with multiplex pcr and clinical applications j clinmicrobiol “ 101128jcm0079307 pmid nakamura y sotozono c kinoshita s the epidermal growth factor receptor egfr role in cornealwound healing and homeostasis exp eye res “ 101006exer2000 pmid fukuoka m yano s giaccone g tamura t nakagawa k douillard jy multiinstitutional randomized phase ii trial of gefitinib for previously treated patients with advanced nonsmallcell lung cancer the ideal trial [corrected] j clin oncol “ 101200jco pmid oldrini b hsieh wy erdjumentbromage h codega p carro ms curielgarcı´a a egfr feedbackinh
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" inflammatory pseudotumour has been used to describe an inflammatory or fibrosing tumoral processof an undetermined cause that may involve a variety of an systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potential for recurrence and persistent local growth in this we report a patient with a big mass of uncertain nature and behaviorcase presentation a 60yearold woman presented with a 1week history of abdominal pain fever and jaundicesix months before she had had right upper quadrant pain that was interpreted as biliary colic a contrastenhancedct scan showed a big mass of soft tissue with diffuse infiltration of the gallbladder displacement of the transversecolon hepatic flexure and duodenum for diagnostic distinction between a chronic inflammatory disease or aneoplasm exploratory laparotomy was required intraoperative exploration disclosed a big mass of hard textureinvolving the gallbladder with multiple concrements hepatoduodenal ligament right and transverse mesocolonstomach and duodenumcholecystectomy was performed preserving adjacent ans with macroscopic desmoplastic reactionhistopathologic examination of the gallbladder showed a spindle cell proliferation with diffuse chronicinflammatory infiltrate of lymphocytes plasma cells and hyalinized fibrous stroma no vascular invasion or cellularatypia were evident inflammatory pseudotumour is a rare condition and diagnostic distinction from a chronicinflammatory disease or other neoplasm is only possible by histopathologic examination there is a limited numberof case reports in the literature indicating tumor location in the gallbladderkeywords inflammatory pseudotumor gallbladder inflammatory pseudotumour is a rare lesion that hasbeen described in various ans and tissues intraabdominal variants of the disease are reported to occurmost frequently in the liver spleen mesentery and extrahepatic bile duct the location of the gallbladder iseven more uncommon correspondence acd3202yahooesdepartment of surgery and pathology puerto real university hospital c¡dizspainmalignant transformations and recurrences of inflammatory pseudotumour have been reported years aftersurgery therefore longterm followup is necessary evenfor patients successfully treated by surgical resectionelevated igg4 serum levels have been reported in association with this illness as well as abundant igg4 positivity in tumor infiltrating plasma cells signs suggestiveof an igg4related disease a high serum igg4 concentrations thus provides a useful means of distinguishingthis disorder from other differential diagnoses 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 0ccalvo bmc gastroenterology page of pharmacologic treatments have also been reported forigg4associated inflammatory pseudotumor and thereare even cases of complete resolution of the disease withsteroids treatments however in the presented case thiscondition did not occur so treatment was exclusivelysurgicalcase presentationa 60yearold woman presented to the emergencyroom with abdominal pain fever pruritus and jaundicesince week the patient had a history of smoking anda family history of pancreatic canceron physical examination a hard and painful mass wasidentified on the right hypochondrium blood laboratoryexamination showed extrahepatic cholestasis enzymestotal bilirubin mgdl direct bilirubin mgdlast ul alt ul ggt ul ldh ul alkaline phosphatase ul in anamnesis thepatient referred to abdominal pain occurring during thelast months located in the right upper quadrant whichhad been interpreted as biliary colic by her generalpractitionertumour markers and blood count showed no alterations viral serology autoimmunity antibodies metanephrines and urine normetanephrine were within thenormal rangea large mass associated with the gallbladder was identified by abdominal ultrasound contrastenhanced ctscan disclosed a large soft tissue mass originating fromthe gallblader with homogenous contrast enhancementand without clear infiltration of the hepatic parenchymathe mass displaced the transverse colon hepatic flexureand duodenum no lymphadenopathies were identifiedin the hepatoduodenal ligament pancreas retroduodenum or celiac axis fig 1a b the gallbladder was distended contained stones and had a regular lumenwhile there was slight dilatation of the intrahepatic bileduct on magnetic resonance imaging fig 1csmallerwith these findings a diagnostic distinction between a chronic inflammatory disease or a neoplasticprocess was necessary the biopsy of the mass wasperformed under ultrasonographic control histopathologic examination showed spindle cells and some inflammatory cells ofsize and absence ofxanthic cells the tumor showed a mesenchymal aspect that was confirmed by absence of epithelial cellson pancytokeratin staining while some histiocyteswere recognized in summary the pathology diagnosiswas a mesenchymal process that could be reactive ormalignant the microbiological study of the bile obtained from gallbladder punctured showed a nonpurulent gram™s stain and negative cultures for bothaerobic and anaerobic germs the oral endoscopy andbiopsies of the second part of the duodenum didn™tshow any pathological conditionexploratory laparotomy was decided and cholecystectomy could be performed preserving the adjacent ans with macroscopic desmoplastic reaction the masswas peeled off the transverse colon first and the secondpart of the duodenum and common bile duct fig the histopathological examination offibrousspecimen disclosed sclerosingthe resectiontissue withfig axial and coronal computed tomography images showing a large mass of diffuse soft tissues originating from the gallbladder anddisplacing the duodenum transverse colon and hepatic flexure a b in magnetic resonance imaging the gallbladder was distended andcontained stones associated with a slight dilatation of the intrahepatic bile duct c 0ccalvo bmc gastroenterology page of stains were performedto achieve a definitive classification complementaryimmunohistochemicalandshowed positive staining for smooth muscle actin in themuscular layer ofthe gallbladder and vessel wallscd34 in the vascular lumen cd68 in histiocytes andremained negative for anaplastic lymphoma kinasealk and pancytokeratin panck masson™s trichrome stain showed intense positivity on collagen fibers less than of the tumor cells sample were ki67positive and plasma cells were igg4 positive per highpower field taken together these findings confirmed thediagnosis of inflammatory pseudotumor of the gallbladder with sclerosing cholangitis associated with a normallevel of serum of immunoglobulin g4 of mgdl “ mgdlno local recurrence was detected at the threeyearsfollowup on ct scandiscussion and sthe term inflammatory pseudotumour has been used todescribe an inflammatory or fibrosing tumoural processof undetermined cause that may involve a variety ofan systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potentialfor recurrence and persistent local growth [ ] thereis a limited number of case reports in the literature indicating gallbladder location [ ]fig on laparotomy a large and hard mass was identified in thegallbladder with stones displacing but not infiltrating right andtransverse mesocolon stomach duodenum andhepatoduodenal ligamenthistiocytes chronic lymphocytic inflammatory infiltrateand plasma cells with isolated eosinophils and no epithelial malignancy the mass presented as an expansivegrowth from the outer portion of the muscular layer ofthe gallbladder to the surrounding fatty tissuethe definitive pathological diagnosis was inflammatorypseudotumour of the gallbladder with chronic sclerosingcholangitis fig a bfig histopathologic examination disclosed a thickened gallbladder wall a with spindle cells and proliferation of connective fibrous tissuewithout signs of celular atypia b and inflammatory cells including lymphocites plasma cells and hyalinized fibrous tissue without vascularinvasion c few plasma cells were igg4 positive in relation to the whole inflammatory cell infiltrate d 0ccalvo bmc gastroenterology page of consent for publicationwe confirm in this statement that a written consent to publish thisinformation was obtained from study participant and the proof of consentto publish from study participants can be provided at any timethe authors have in their possession the informed consent of the patientbiomed central consent formcompeting intereststhe authors declare that they have no competing interestsreceived january accepted august referencesbehranwala ka straker p wan a fisher c thompson jn inflammatorymyofibroblastic tumour of the gallbladder world j surg oncol sinha l hasan a sngh ak bhadani pp jha an singh pk kumar minflammatory myofibroblastic tumor involving liver gallbladder pylorus andduodenum a rare case presentation int j surg case rep “badea r veres aa andreica v inflammatory myofibroblastic tumor ofthe gallbladder imaging aspects j med ultrason “abrantes cf silva mr oliveira rc eloy c cipriano ma castro lpinflammatory myofibroblastic tumour arising incidentally as a polypoidlesion in the gallbladder j bras patol med lab v51 n p “koea jb broadhurst gw rodgers ms inflammatory pseudotumor ofthe liver demographics diagnosis and the case for nonoperativemanegement j am coll surg “sato y kojima m takata k immunoglobulin g4relatedlymphadenopathy with inflammatory pseudotumorlike features med molmorphol “hamano h kawa s horiuchi a high serum igg4 concentrations inpatients with sclerosing pancreatitis n engl j med “aldhahab h mcnabbbaltar j albusafi s barkun an immunoglobulin g4related pancreatic and biliary disease can j gastroenterol “lee ys lee sh lee mg immunoglobulin g4related diseasemimicking unresectable gallbladder c¡ncer gut liver “ muduly d deo sv shukla nk inflammatory myofibroblastic tumor ofgall bladder trop gastroenterol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsinflammatory pseudotumour appear to be more common in noneuropean populations they usually occur ininfancy and young adults but can occur in the elderly []the igg4 serum level should be determined due to acommon association of elevated serum igg4 with this illness and some authors describe abundant igg4 positivityin plasma cells as suggestive of igg4related disease []high serum igg4 concentrations might provide afromuseful means of distinguishing this disorderother lesions [“]the histopathological examinations showed sclerosingfibrous tissue with histiocytes chronic lymphocytic inflammatory infiltrate and plasma cells with isolated eosinophils compatible with igg4related disease howeveras igg4 serum levels were within normal range and theigg4 tissue expression was very weak there is no strongevidence of a clear association with igg4related diseaseinflammatory pseudotumour can generally be considered to be a relatively rare disease of undefined originwith a great variety of symptoms causing diagnosticchallenges in the distinction of chronic inflammatorydisease and neoplasminflammatory pseudotumoris defined as nonneoplastic but is currently considered as a tumour withlowgrade malignant transformations it has been reported in the liver urinary bladder kidney breast stomach pancreas spleen and retroperitoneum there is alsoa limited number of case reports in the literature indicating the gallbladder location these patients must beobserved with close and regular longterm followup asrecurrences have been reported to occur four to yearsafter surgery []acknowledgementsangela hens head of the pathology department of puerto real universityhospital for providing her pathological knowledgemario bruno for providing the new corrections in the translation of thismanuscriptpd dr med ulrich f wellner consultant surgeon pancreatic surgery andresearch clinic of surgery uksh campus l¼beck germany for providingthe latest english language correctionsauthors™ contributionsac the first author directed the operation and wrote the paper js hasparticiped in the design of the report and copy edited the manuscript adhas participated in the operation mc has participated in the operation gmhas made the pathological diagnosis and part of the literature review allauthors read and approved the final version of the manuscript all authors ofthis manuscript are in agreement with its content and are not beingpublished or under consideration in another scientific journalfundingnot applicableavailability of data and materialsdata sharing is not applicable to this as no data sets were generatedor analysed during the current studyethics approval and consent to participatenot applicable 0c"
0
"annual meeting of the european associationfor the study of diabetes“ september sindex of oral presentationsop diabetes complications new insights from cutting edge epidemiologyop news on the insulin secretion frontop insulin sensitivity and biomarkersop central actions in diabetesop glucoselowering therapies and the liverop uncomplicating the pathogenesis of diabetes complications inhumansop smoke on the water is bat still hotop charting human beta cell failure in type diabetesop novel agents in type diabetesop developing better insulinsop from diagnostics to the endstage of diabetic kidney diseaseop nafld is it all about the liverop diabetic retinopathy see what's newop taking the long view of diabetesop pregnancy in diabetes prediction and outcomesop signals and networks in beta cell failureop broken heart in diabetesop unlocking the potential of digital healthop decoding the heritable basis of type diabetesop feeding the pipeline from drugs to surgeryop sglt2 inhibitors at the heart of the matterop new treatments for nafld hope or hypeop addressing potential new treatments of diabetic kidney diseaseop glucagon and hormones beyondop incretin based therapiesop unusual forms of diabetesop macrovascular complications and beyondop linking inflammation to metabolismop what's new in automated insulin deliveryop understanding the mechanisms of diabetic kidney diseaseop novel aspects of diabetic neuropathyop reducing the burden of hypoglycaemiaop what exercise doesop back to the future risk markers in diabetesop diet not only quantity mattersop on the road to human islet failure in type diabetesop a deep dive into the mechanisms of diabetesop triggers and drivers of beta cell failure in type diabetesop gastroentero pancreatic factors anoids mice and menop new aspects of novel therapiesop fatty mattersop diabetes care is expensiveop developing beta cellsop modelling metabolism lessons from animalsop diabetic foot new developments in wound healingop challenges in delivering diabetes care new solutionsop thinking about diabetes complications in the brainop insulin secretion in various subgroupsindex of poster sessionsps diabetes and early deathps living with chronic diabetes complicationsps micro and macrovascular complications of diabetesps global view on diabetes complicationsps type diabetes treatment irlps unusual forms of diabetesps molecular insights into glucose abnormalitiesps pathophysiology of glucose homeostasisps the inner workings of the pancreasps islets and antibodies in type diabetesps markers and phenotypes of glucose traitsps global aspects on the epidemiology of type diabetesps risk factors for type diabetesps prevalence of type diabetes around the worldps risk factors in type diabetesps islet transplants revisitedps islets in type diabetes new playersps beta cells under stressps to live and let die a beta cell perspectiveps job description insulin secretionps further down the road to human islet failure in type diabetesps sitting and exercising does it allps the ins and outs of carbohydrate metabolismps pregnancy in vitro and in vivo studiesps pregnancy epidemiologyps pregnancy who is at riskps incremental studies on gut hormonesps the fundamentals of insulin resistanceps studies on insulin resistanceps treatment of hyperglycaemia in pregnancyps pancreatic hormonesps insulin secretion in mice and menps something more about obesityps more about metabolismps inflammation in type diabetesps models of prediabetes and diabetesps models of obesity and insulin resistanceps lipid metabolismps adipokine signallingps drugs and environment in obesityps weight loss interventionsps brain mattersps sglt2 inhibitors clinical aspectsps different aspects of sglt2 inhibitorsps basic aspects of incretinbased therapiesps clinical outcome of incretinbased therapies 0cdiabetologiaps glycaemic control and incretinbased therapiesps various clinical aspects of incretinbased therapiesps various aspects of nutrition and dietps oral therapies metformin sensitizers and other nonsecretagoguesps novel agents to treat diabetes and its consequencesps novel glucoselowering agents in type diabetesps key issues in improving outcomes in people with diabeteseducation and costsps how to improve diabetes careps the impact of new basal insulinsps insulin therapy real world studiesps insulin therapy fast acting insulin analoguesps the challenges of insulin therapy in type diabetesps different aspects of insulin therapyps the continued advance of continuous glucose monitoringps insulin pump therapyps automated insulin deliveryps the varied use of technologies in type diabetesps novel applications of technology in diabetesps novel therapies to reduce hypoglycaemiaps mechanisms and clinical consequences of hypoglycaemiain diabetesps emerging topics in hypoglycaemiaps investigating diabetes distress and depressionps aspects of quality of life and well beingps digital health in type diabetesps is telehealth the answer to improving care in diabetesps predicting prognosis of diabetic kidney diseaseps clinical aspects of diabetic kidney diseaseps the rock and role of experimental kidney diseaseps new tools to view diabetic retinopathyps diabetic retinopathy screening and interventionps focus on diabetic foot ulcersps hypertension and vascular diseaseps cure the pain of diabetic neuropathyps understanding clinical neuropathyps from artificial intelligence to treatment of diabetic footps from biomarkers to genetics of diabetic kidney diseaseps treatment of nafld and diabetes from food to pharmacologyps mechanisms and prevalence of nafldps lipids everywhere lipid metabolism in the liver and the heartps all about coronary arteries and diabetesps lipids and glucose not so good for the heartps cardiac complications of mice rats and cellsps atherosclerotic complications stemming from cells to the kidneyps stiff arteries and how to avoid themps cardiac function and dysfunctionps cardiovascular complications in humans through and throughps diabetes and neoplasiaps contemplating cognitive dysfunction in diabetesps endothelial cell circulation and the heartps tradition no nontraditional complications of diabetes 0cdiabetologiaop diabetes complications new insightsfrom cutting edge epidemiologycirculating metabolites significantly improve the prediction of renaldysfunction in type diabetesm scarale1 s de cosmo1 c prehn2 f schena3 j adamski2 vtrischitta4 c menzaghi11fondazione irccs œcasa sollievo della sofferenza san giovannirotondo italy 2helmholtz zentrum m¼nchen germany 3universityof bari bari italy 4sapienza university roma italy and aims chronic kidney disease ckd mainly indicated by a reduced glomerular filtration rate gfr remains one of theleading causes of reduced lifespan in patients with type diabetest2d discovering novel biomarkers able to predict low gfr will helpidentify highrisk patients to be targeted to more aggressive and burdensome preventive and treatment strategiesmaterials and methods we measured serum metabolites byabsoluteidqtm p180 kit biocrates life sciences ag innsbruckaustria and investigated their association with egfr calculated with theckdepi formula in a discovery sample of patients with t2d cases and controls with egfr60 and ‰¥70mlmin173m2 respectively a threshold p value of 28x104 ie followingbonferroni's rule was used as statistical significance in a model comprising age sex smoking bmi hba1c diabetes duration albumintocreatinine ratio acr and ongoing treatments metabolites associatedin the discovery sample were validated threshold p value of 005numberof surviving validation metabolites in a second cohort comprising diabetic patients cases and controls for egfr60 or ‰¥70mlmin173m2 respectively standardized values of each validated metabolitesweighted for the effect size ie observed in the discovery samplewere then summed up in a metabolic score metscore to be used as agfr prediction tool to this purpose metscore was used on top of anestablished clinical model comprising sex age bmi hba1c and acrand then discrimination [δ area under the receiver operating characteristic roc curve auc and the relative integrated discriminationimprovement ridi] and reclassification [the categoryfree net reclassification index cnri] measures were evaluatedresults thirteen metabolites six acylcarnitines six biogenic amines andone amino acid were independently associated to low egfr [ors range for 1sd increase p range 13x107 20x104] in the discoverysample all of them but one a biogenic amine were validated in thereplication sample [ors range for 1sd increase p range32x1018 43x106 below the threshold of 0051242x103] theauc of the abovementioned clinical model was and in the discovery the replication and the pooled sample respectively the addition of metscore on top of the clinical model improvedboth discrimination and reclassification measures in the discovery δauc4 p14x103 ridi29 p20x1011 ½cnri54p15x1014 the replication δ auc39 p16x103 ridi28p38x108 ½cnri30 p22x1010 and the pooled samples δauc39 p40x106 ridi29 p22x1017 ½cnri35p19x108conclusion we have discovered and validated metabolites that arestrongly associated with low egfr in patients with t2d a metscorecomprising these metabolites improves an established clinical prediction model of low egfr in terms of both discrimination and reclassification encouraged by these findings we are now investigating the ability ofmetscore to improve prediction of gfr decline in prospective cohorts oft2d with the aim of improving risk stratification and therefore refiningprevention efforts of kidney dysfunction in diabetic patientssupported by italian ministry of health rf201302356459disclosure m scarale noneassociation between insulinlike growth factor binding protein2 andinsulin sensitivity metformin and mortality in patients with newlydiagnosed type diabetesmr kristiansen12 js nielsen12 i brandslund3 da olsen3 jvstidsen2 sk nicolaisen4 r hjortebjerg25 j frystyk561danish centre for strategic research in type diabetes dd2odense 2steno diabetes center odense odense 3irs lillebaelthospital biochemistry and immunology vejle 4department ofclinical epidemiology aarhus 5department of clinical researchuniversity of southern denmark odense 6department ofendocrinology odense university hospital odense denmark and aims insulinlike growth factor binding protein2igfbp2 is engaged in metabolism circulating concentrations ofigfbp2 are positively correlated to insulin sensitivity overexpressionof igfbp2 protects against obesity and diabetes in mice and metforminincreases igfbp2 gene expression indicating that igfbp2 is a target ofmetformin action interestingly igfbp2 appears to predict mortalityindependently of insulin sensitivity this study aimed to investigate theassociation between indices of insulin sensitivity metformin treatmentand mortality in patients with newly diagnosed type diabetes t2dmaterials and methods in this crosssectional study we included newlydiagnosed patients with t2d enrolled in the danish centre for strategicresearch in type diabetes dd2 cohort patients were continuouslyenrolled from to throughout denmark and followed usingdanish healthcare registries unbound fractions of igfbp2 were determinedin serum from fasting drug na¯ve n864 and metformin treated ‰¥ twoprescriptions months prior enrollment patients n558 using an inhouseassay developed on the simoa platform values are given as medians iqrassociation was analyzed using a pearson™s regressioncox regression amultivariable model was used to adjust for age bmi and homasresults a total of patients with median age of medianbmi of and median diabetes duration of yearswere included igfbp2 level was positively correlated with homasr2026 and p0005 and inversely correlated with cpeptide r2018and p0005 both associations persisted following adjustments for ageand bmi the igfbp2 level in metformin treated patients was slightlylower ngml than in drug na¯ve patients ngml p0026 a total of patients suffered from one or morecomorbidities from charlson comorbidity index their igfbp2 levelswere higher than patients with no comorbidity vs ngml p0001 during a median of years offollowup a total of patients died igfbp2 level was significantly higher at baseline in patients that died vs not died vs ngml p0001 igfbp2 was associated withmortality with a hazard ratiohr ci per doubling in proteinconcentration of p0001 this association was notobserved when analyzing patients without comorbidities but was significant in patients with other comorbidities hr p0001conclusion this is the first larger study to confirm that igfbp2 isassociated with indices of insulin sensitivity but is not largely affectedby metformin treatment interestingly increased igfbp2 level is associated with high mortality rates but the association was mainly driven bythe presence of comorbidities at baselinesupported by university of southern denmark and region of southerndenmarkdisclosure mr kristiansen nonebuilding clinical risk score systems for predicting allcause andcardiovascularspecific mortality among type diabetes patientscs liu1 tc li2 cc lin1 ci li11china medical university hospital taichung 2china medicaluniversity taichung taiwan 0c and aims no prior prediction model for mortality considered the effect of glycemic variability and blood pressure variabilitywhich have been broadly reported as the important clinical predictors ofmortality especially in diabetes patients the aim of this study was todevelop and validate risk score systems with considering the effects ofglycemic and blood pressure variability on allcause and cardiovascularspecific mortality in persons with type dmmaterials and methods this is a retrospective cohort study consistingof type diabetic patients aged years during allparticipants were randomly allocated into two groups derivation andvalidation sets in ratio and were followed up until death or august cox proportional hazards regression were used to develop allcauseand cardiovascularspecific mortality prediction model prediction modelperformance was assessed by the area under the receiver operating characteristics curve aurocresults overall deaths were identified after a mean of years offollowup the prediction accuracy measured by auroc of and 15year allcause mortality based on a model containing the identifiedtraditional risk factor biomarkers and variability in fasting plasmaglucose and hba1c and diastolic blood pressure variability were and respectively in derivation set and the corresponding values forcardiovascularspecific mortality were and respectively the predictionaccuracy in the validation set for allcause mortality were and respectively and for cardiovascularspecific mortality were and respectivelyconclusion our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascularspecific and allcause mortality in patients with type diabetessupported by ministry of science and technology of taiwandisclosure c liu noneincident cardiovascular disease by clustering of favourable riskfactors in type diabetes the eurodiab prospectivecomplications studys soulimane1 yd vogtschmidt12 m toeller3 b balkau4 nchaturvedi5 jh fuller6 ss soedamahmuthu121department of medical and clinical psychology center of research onpsychological and somatic disorders corps tilburg universitynetherlands 2institute for food nutrition and health university ofreading reading uk 3heinrichheineuniversity d¼sseldorfd¼sseldorf germany 4clinical epidemiology universit© parissaclayuvsq inserm cesp villejuif france 5institute of cardiovascularscience university college of london london uk 6department ofepidemiology and public health eurodiab london uk and aims the incidence of cardiovascular diseases cvdis up to eight times higher in people with type diabetes t1d greaterclustering of adverse risk factors is thought to contribute to excess cvdrisks in type diabetes though not explored in t1d the aim of this studywas to examine a cvd risk reduction for those in the most favourablethird of individual risk factors compared to the least favourable two thirdsand b cvd risk reduction by clustering of favourable cvd risk factorsmaterials and methods we analysed data of participants from theeurodiab prospective complications study a european t1d cohortrecruited in countries between were men with a meanage of ± years we studied seven cvd risk factors namely hba1csmoking bmi combined systolic and diastolic bp ldl cholesterolphysical activity pa and diet table cox proportional hazards analyses were used to calculate hazard ratios hr [95ci] of incident cvdfor each cvd risk factor adjusted for age sex retinopathy comparingthose in the most favourable tertiles with the least favourable two tertilesdiabetologiawe then scored each individual by the number of risk factors for whichthey occupied the most favourable tertilesresults there were incident cvd cases after a mean followup of± years multivariable cox models showed that participants withthe most favourable hba1c57 [39mmolmol] had a significantlylower cvd risk hr [95ci] [] than the least favourabletwo tertiles nonsignificant inverse associations were found withfavourable bmi [] pa [] diet score[] and bp [] no associations were foundwith smoking or ldlcholesterol greater clustering of favourablecvd risk factors was associated with a lower risk of cvd in univariatemodels with a significant linear trend in multivariate models the resultswere partly attenuated with the lowest hr of [ ] in peoplewith clustering of favourable cvd risk factors tableconclusion greater clustering of favourable cvd risk factors was associated with a lower risk of incident cvd in people with t1d with a doseresponse relationship hba1c remained the most protective factor againstcvd in t1d targeting combined risk factors could be more effective inpreventing cvd risk than targeting single risk factorssupported by welcome trust the european community and diabetesukdisclosure s soulimane nonebidirectional association between type diabetes and obstructivesleep apnoea a metaepidemiological studyt karagiannis1 e athanasiadou1 a tsapas12 e bekiari11clinical research and evidencebased medicine unit aristotleuniversity of thessaloniki thessaloniki greece 2harris manchestercollege university of oxford oxford uk and aims individual epidemiological studies suggest acomplex relationship between type diabetes and obstructive sleepapnea we aimed to assess whether there is a bidirectional associationbetween the two conditions by conducting a metaanalysis of longitudinalcohort studiesmaterials and methods we included cohort studies that evaluated theassociation between type diabetes and obstructive sleep apnea in eitherdirection published until january we pooled cohortspecific estimates by means of random and fixed effects metaanalyses and calculatedodds ratios ors with confidence intervals cis to measure theassociation of prevalent obstructive sleep apnea with incident type diabetes and of prevalent type diabetes with incident obstructive sleepapnearesults out of records identified through the search cohortstudies were included in the metaepidemiological analysis ten studiesevaluated the association between prevalent obstructive sleep apnea andincident type diabetes one study assessed the association betweenprevalent type diabetes and incident obstructive sleep apnea while fourstudies evaluated a bidirectional association duration of study followupranged between and years median years the random effectsmetaanalysis for prevalent obstructive sleep apnea and incident type diabetes patients yielded an or of ci to 0cdiabetologiaresults were consistent in the fixed effects metaanalysis figureprevalent type diabetes increased the odds of incident obstructive sleepapnea patients with an or of ci to and ci to for the randomeffects and fixedeffects metaanalysis respectively metaanalyses of effect estimates adjusted forconfounding factors were similar to those of the main analysisconclusion pooled evidence from large cohort studies suggests thatpresence of obstructive sleep apnea at baseline is associated withincreased risk for developing type diabetes while presence of type diabetes is associated with increased risk for developing obstructive sleepapnea thus effective management of either condition could preventdevelopment of the otherfigure odds ratio for developing type diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apneaalzheimer hr [ ic ] vascular dementia hr [ ic ] and nonvascular dementia hr [ ic ] when a 3years landmark analysis was conducted the associations remained similar for vascular and nonvascular dementia but disappeared for alzheimer™s diseasesconclusion the association of t2d with neurodegenerative diseasesdiffer by type of dementia the strongest detrimental association wasobserved for vascular dementia moreover t2d patients with polycaemic control have an increased risk of developing vascular andnonvascular dementiadisclosure c celismorales nonesupported by greece and the european social fund esfdisclosure t karagiannis noneglycated haemoglobin type diabetes and the links to dementia andits major sub types findings from the swedish national diabetesregisterc celismorales1 s franz©n2 am svensson3 n sattar1 sgudbjornsdottir21institute of cardiovascular and medical sciences university ofglasgow glasgow uk 2department of molecular and clinicalmedicine university of gothenburg gothenburg sweden 3swedishnational diabetes register gothenburg sweden and aims type diabetes t2d has been associated withhigh dementia risk however the links to different dementia subtypes isunclear we examined to what extent t2d associated with alzheimervascular and non vascular dementia incidence and whether such associations differed by glycaemic controlmaterials and methods in this swedish national diabetes registerstudy we included patients with t2d and matchedcontrols the outcomes were incidence of alzheimer vascular and nonvascular dementia the association of t2d with dementia was stratifiedby baseline glycated haemoglobin hba1c concentrations cox regression was used to study the excess risk of outcomesresults the followup median years t2d patientsand controls developed dementia the strongest association was observed for vascular dementia here patients with t2d had ahr of [ ci ] compared to controls the association oft2d with nonvascular dementia was more modest hr [ ci ] however risk of alzheimer was lower in t2d patientscompared to controls hr [ ci ] when the analyseswere stratified by circulating concentrations of hba1c a doseresponseassociation was observed compare to patients with t2d with hba1c mmolmol those with hba1c mmolmol had a higher risk of 0cop news on the insulin secretion frontwhat makes beta cells 1st responders and are they temporallyconsistentv kravets we schleicher jm dwulet am davis rkpbenningerbioengineering university of colorado aurora usa and aims calcium ca2 uptake drives glucosestimulated insulin secretion from the pancreatic cells functionalsubpopulations of cells disproportionally control the oscillatory phaseof ca2 uptake which is disrupted with ageing and in diabetes less isknown about cells which impact the 1st phase of ca2 uptake disruptedin early diabetes here we determine whether œ1stresponder cells thatlead the 1st phase of ca2 uptake are the same as œhub cells that coordinate oscillatory ca2 2nd phase we study what makes cell a1st responder and whether 1st responders are a transient state or a distincttemporally stable subpopulationmaterials and methods we used mipcreer gcamp6s mouse modelwhich expresses ca2sensitive gfp specifically in cells weperformed simultaneous recording of ca2 dynamics and gap junctionpermeability in individual islets we stimulated islets with glucosekatp channel blocker glibenclamide and kcl based on ca2 dynamicswe defined the of cells responding to the glucose stimulation soonerthan the rest of the islet as œ1st responders and the of cellsresponding slower as œlast responders we tested their temporal consistency over and hours we used laser ablation to remove specificcells from the islet we performed computational modelling of the isletelectrophysiologyresults we found that ca2 wave coordination of the 1st responders wasnot greater than the isletaverage and hence they are not overlapping withhighlycoordinated œhub cells in fact according to our gap junctionpermeability data 1st responders had lower than average electricalcoupling p00157 furthermore our computational model showedlower electrical coupling conductance in both 1st and last respondersp00447 p00279 this may be explained by our finding that1st responders are located at the islet™s periphery at ± of the islet™sradius we found 1st responders to be consistent under glibenclamidestimulation cells which respond first to the glucose remained in the15th percentile of the time response distribution when stimulated withglibenclamide sem this is consistent with our computationalresults 1st responders had lower katp conductance hence highermembrane depolarization probability p00086 glucose elevationswith 1h period showed that 1st responders remained consistent withreaction time within the of the reaction time distribution for all cellswith an elevation period of hours their reaction time shifted to thesecond quartile of the distribution and with hours to the medianunlike 1st responders last responder cells were not consistent at any timeinterval ablation of the 1st responders discoordinated but did notdisrupt the ca2 response of the islet a different cell took over the roleof the 1st responder postablation this new 1st responder was a cell whichoriginally preablation was within a leading 7th percentile of the timeresponse distribution sem conclusion in conclusion 1st responders are distinct from œhub cellsubpopulation have higher membrane depolarization probability and areless strongly coupled to other cells after the laser ablation of a1st responder new 1st responder taking on it™s role comes from a poolof original leading cells while initially consistent over a short 1h periodof time 1st responders may be losing temporal consistency over longertime periodssupported by nr01 dk102950 dk106412 jdrf 3pdf2019741andisclosure v kravets nonediabetologiabetaarrestin is absolutely required for the potentiation of insulinsecretion by gipma ravier1 j obeid1 m leduc1 s costes1 p gilon2 s dalle1 gbertrand11igf univ montpellier cnrs inserm montpellierfrance 2universit© catholique de louvain brussels belgium and aims the scaffold protein betaarrestin2 arrb2 isknown to uncouple g protein coupled receptors gpcrs from the gprotein and to recruit new signaling pathways such as the erk12pi3k fak‹¯ in non beta cells arrb2 interacts with a wide rangeof gpcrs but its interaction with the gip receptor gipr is still unclearour aim is to determine if arrb2 is involved in the signaling of thegipr in pancreatic beta cellsmaterials and methods the experiments were carried out in beta cellsfrom fivemonthold arrb2 and arrb2 male mice camp productioncampsepac endogenous pka akar3 and erk12 ekaractivations [ca2] in the cytosol [ca2]c fura2lr and in the endoplasmic reticulum [ca2]er d4er were assessed by live cell imagingin mouse pancreatic beta cells epac2 epac2gfp recruitmentbeneath the plasma membrane was monitored by total internal reflectionfluorescence microscopy factin depolymerisation was evaluated byphalloidin staining alexa fluor 488conjugated phalloidin and thep h o s p h o r y l a t i o n o f f o c a l a d h e s i o n k i n a s e f a k b yimmunofluorescenceresults insulin secretion from arrb2 islets was reduced by compared to arrb2 islets in response to gip 100pm10nm p001when arrb2 arrb2gfp was reexpressed in arrb2 beta cells insulin secretion in response to gip was restored to a similar level thanin arrb2 islets surprisingly upon gip stimulation 10pm10nm thecamp production pka activation and epac2 recruitment were similarin arrb2and arrb2 beta cells both [ca2]c and [ca2]er remainedcomparable finally the activation of erk12 was also similarin arrb2 and arrb2 beta cells by contrast the factin depolymerisationinduced by 10nm gip was significantly reduced p001 in arrb2 beta cells pi3kγ and fak have been reported to be involved in factindepolymerisation in response to gip and glucose respectively and to berequired for optimal insulin secretion as expected the pi3kγ inhibitoras604850 1μmoll reduced factin depolymerisation p001by gip stimulation in arrb2 beta cells but no additional effect wasobserved in arrb2 beta cells moreover gipinduced fak activationwas also reduced by in arrb2 beta cellsconclusion our study revealed that arrb2 is required for the potentiation of insulin secretion by gip through factin depolymerisation probably via fak activation and pi3kγ recruitment but independently fromthe canonical camp signalling pka and epac2 and the erk12 pathway therefore any variation in the expression of arrb2 as observed indiabetic states should functionally affect the incretin effect produced bygipsupported by soci©t© francophone du diabete sfddisclosure ma ravier nonepancreatic beta cellselective deletion of the mitofusins and mfn1and mfn2 impairs glucosestimulated insulin secretion in vitro andin vivoga rutter1 e geiadou1 t rodriguez2 c muralidharan3 mmartinez3 p chabosseau1 a tomas1 g carrat1 a di gregorio2 ileclerc1 ak linnemann31cell biology functional genomics faculty of medicine imperialcollege london london uk 2national heart and lung instituteimperial college london london uk 3center for diabetes andmetabolic diseases indiana university school of medicineindianapolis usa 0cdiabetologia and aims mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cellsalthough altered in subjects with type diabetes whether mitochondrialultrastructure and the proteins controlling the fission and fusion of theseanelles are important for glucose recognition is unclear here wegenerated mice with beta cellselective adultrestricted deletionof mfn1 and mfn2 essential for mitochondrial fusion and studied theimpact on insulin secretion and glucose homeostasis in vivo and in vitromaterials and methods c57bl6 mice bearing mfn1 and mfn2 alleleswith floxp sites were crossed to transgenic animals carrying aninducible cre recombinase under pdx1 promoter control pdxcreertrecombination was achieved by daily tamoxifen injections for one weekislets were isolated and used for live beta cell fluorescence imaging ofcytosolic cal520 or mitochondrial rgeco free ca2 concentrationand membrane potential tetramethyl rhodamine methyl ester tmrmusing spinning disc confocal microscopy nikon ti2 mitochondrialnetwork characteristics were quantified using super resolution fluorescence zeiss lsm and transmission electron microscopy intravitalimaging was performed in mice injected with an adenoassociated virusto express the cytosolic ca2 sensor gcamp6s selectively in beta cellsunder the control of the rat insulin promoter using multiphoton microscopy leica tcs sp8 dive blood flow through the islet was visualisedsimultaneously after injection of fluorescent albumin647results mitochondrial length was sharply to ± of controlsp00001 reduced in the mfn12 ko mice and these animals displayedhigher fasting glycaemia than control littermates at weeks vs mmoll p005 in vivo an increase in circulating glucose levelswas also observed p005 at min and p001 at min and wasassociated with a substantial fivefold decrease in plasma insulin min p00001 postintraperitoneal glucose injection mitochondrialca2 accumulation and membrane potential were significantly reducedp001 in response to high glucose in the ko animals examined byintravital imaging of the exteriorised pancreas antiparallel changes incytosolic ca2 and mitochondrial membrane potential observed incontrol animals were largely suppressed after mfn12 deletionconclusion mitochondrial fusion and fission cycles are essential in thebeta cell to maintain normal mitochondrial bioenergetics and glucosesensing both in vitro and in the living mouse such cycles may bedisrupted in some forms of diabetes to impair mitochondrial functionand consequently insulin secretio
0
"decreased expression of Hsp70 caused by ibuprofen amplified the activation of caspase-9 significantly compared with that of Bax. Furthermore the similar increase in the activation of cisplatin-dependent Bax and release of cytochrome c by ibuprofen suggests that Hsp70 also inhibits the post mitochondrial steps between the release of cytochrome c and the activation of caspase-9. If Hsp70 were acting downstream of the mitochondria one would predict that it interferes with the activation of caspase-9 in response to cytochrome c either by inhibiting the formation of the apoptosome or by preventing the binding of pro-caspase-9 to this complex. When we studied the effects of Hsp70 on the formation of and recruitment of pro-caspase-9 to the apoptosome the cell lysates were immunoprecipitated although Hsp70 failed to migrate with Apaf-1 cytochrome c or caspase-9 (data not shown). These results may be supported by previous report that no association between Hsp70 and Apaf-1 or apoptosome complex was observed even under in vitro activation of caspase by the addition of cytochrome c and dATP.44 Furthermore we were unable to identify a new target for Hsp70 in the process of caspase-9 activation indicating that its inhibitory activity is attributable to another indirect effect instead of a direct one as previously reported. Altogether the data presented here are the first evidence of cell death inhibition by Hsp70 by its targeting of both upstream and downstream mitochondrial processes while the precise mechanisms by which it interferes with the activation of caspase-9 remains to be clarified. In ibuprofen potentiated the antitumoural properties of cisplatin in the cells of lung adenocarcinoma via a mechanism of action mediated by the suppression of Hsp70. These findings may promote the development of a new strategy to increase the effectiveness of cisplatin in the treatment of NSCLCs as well as highlight the putative merits of developing anticancer treatments targeting Hsp70. Materials and Methods Materials The mouse monoclonal anti-Hsp70 the rat monoclonal anti-Hsc70 and rabbit polyclonal HSF-1 antibodies purchased from Stressgen “ Enzo Life Sciences Inc. Plymouth Meeting PA USA. Anti-Bax rabbit polyclonal (N-20) and anti-VDAC-1 goat polyclonal (N-18) antibodies were purchased from Santa Cruz Biotechnology Inc. Santa Cruz CA USA. Anti-cytochrome c mouse monoclonal antibody (556433) was obtained from BD Pharmingen Inc. San Diego CA USA. Anti-caspase 9 and -ERK antibodies were acquired from Cell Signaling Technology Inc. Danvers MA USA. The mouse monoclonal antibody against actin was obtained from Chemicon International Inc. Temecula CA USA. Anti-Bax 6A7 monoclonal antibody and other reagents were purchased from Sigma-Aldrich St. Louis MO USA. Cell culture and viability assay A549 and H358 lung cancer cell lines were cultured in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum at 37?°C. BEAS-2B cells were grown in bronchial epithelial basal medium. All NSAID and cisplatin were dissolved in dimethyl sulphoxide and added to the medium at indicated concentrations. The activity of mitochondrial dehydrogenase 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) assay was used to measure cell death/survival. The reaction product was measured at A570 and the relative viability of cells treated with reagents versus untreated cells was calculated. TUNEL staining The TUNEL assay was performed using an in situ cell death detection kit (F Hoffmann-La Roche Basel Switzerland) according to the manufacturer's instructions. The ratio of TUNEL-positive cells to the total number of cells was calculated. Immunoprecipitation and cell fractionation A549 cells were lysed in RIPA buffer (50?mM Tris-HCl pH 7.5 150?mM NaCl 1?mM sodium orthovanadate 1?mM EDTA 0.1% NP-40 10?mM NaF) containing the Calbiochem Protease Inhibitor Cocktail Set III (Merck KGaA Darmstadt Germany). The cell lysates and immunoprecipitates were resolved in Laemmli sample buffer. The samples underwent sodium dodecyl sulphate-polyacrylamide gel electrophoresis were transferred to a polyvinylidene difluoride membrane reacted with the respective antibodies and detected with an ECL chemiluminescence detection kit (GE Healthcare Fairfield CT USA). For the immunoprecipitation the cell lysates were incubated with the indicated antibodies for 1?h at 4?°C. Protein G-sepharose beads were added to collect the immunocomplexes for an additional 1?h of incubation. The pellets were washed three times with lysis buffer. The mitochondria and cytosol fractions were prepared as described previously.45 Chromatin immunoprecipitation (ChIP) assay ChIP assays were performed as described previously46 using an EZ ChIP kit (Upstate Biotechnology Inc. Waltham MA USA). Briefly after adding formaldehyde A549 cells were suspended in SDS lysis buffer and the chromatin DNA was disrupted by sonication. For the immunoprecipitation the lysate was incubated with anti-HSF-1 antibody followed by immobilization on salmon sperm DNA/Protein G agarose. The protein/DNA complexes extracted with elution buffer were heated to 65?°C for 6?h to reverse cross-links then digested with proteinase K. DNA fragments were amplified in PCR with the ChIP assay primers containing the heat shock element sites in human Hsp70 promoter. PCR primers for the ChIP assay were as follows: Hsp70 (?103/+7) (F) 5?-TGATTGGTCCAAGGAAGGCT-3? and (R) 5?-AAAAAGGTAGTGGACTGTCGC-3?. Reverse transcriptase-PCR RT-PCR was carried out using a Qiagen (Valencia CA USA) One-Step RT-PCR Kit. We used the following primer pairs to amplify. Hsp70 (F) 5?-ATGAAGCACTGGCCTTTCCA-3? (R) 5?-TTGTTCTGGCTGATGTCCTT-3? Hsc70 (F) 5?-TGGAACTATTGCTGGTCTCAA3? (R) 5?-AGAACCACCAACCAGGACAAT-3? HSF-1 (F) 5?-TTCGACCAGGGCCAGTTT-3? (R) 5?-AGAGCTGGCCACAGCATCA-3? actin (F) 5?-AGAGGCATCCTCACCCTGA-3? (R) 5?-CATCTCTTGCTCGAAGTCCA-3?. The products were examined by agarose gel electrophoresis after 23 cycles. RNA interference The sequences of the sense strands used to generate specific siRNA were obtained as follows: HSF-1 5?-AAGTACTTCAAGCACAACAA-3? 5?-AAGAGTGAAGACATAAAGAT-3? 5?-AAGTCGTCAACAAGCTCATT-3?. The siRNAs were synthesized using the Silencer siRNA construction kit (Ambion; Applied Biosystems Inc. Carlsbad CA USA). Double-stranded Hsp70 and control siRNA duplex were synthesized as followed by Qiagen: Hsp70-specific sequence 5?-CCAUUGAGGAGGUAGAUUAdTdT-3?. A549 cells were transfected with each siRNA (10?nmol/l) using the Lipofectamine 2000 (Invitrogen; Applied Biosystems Inc.) and grown for 72?h to allow an effective decrease in the expression of the respective target molecules. Quantification of apoptosis by flow cytometry A549 cells were washed with Annexin V staining buffer (10?mM HEPES pH 7.4 150?mM NaCl 5?mM KCl 1?mM MgCl2 1.8?mM CaCl2) and incubated with CF488A-Annexin V and propidium iodide (Biotium Inc. Hayward CA USA) in staining buffer for 30?min at 37?°C in the dark. Fluorescence was measured using a FACSCalibur (BD Biosciences San Jose CA USA) and the data were analyzed with CellQuest software (BD Biosciences). JC-1 staining and quantification A549 cells were cultured at 37?°C for 48?h on glass chamber slides and treated with ibuprofen and cisplatin at the specified concentrations for 48?h. Mitochondrial permeability transition was determined by staining the cells with 55?66?-tetrachloro-1133?-tetraethyl- benzimidazolylcarbocyanin iodide (JC-1; Molecular Probes Invitrogen Carlsbad CA USA) in the dark. The cells were subsequently washed with assay buffer according to the manufacturer's protocol and immediately imaged using a fluorescence microscope (Keyence Corporation Osaka Japan) with the red (?excitation: 560±40?nm band pass filter ?detection: 630±60?nm band pass filter) and green (?excitation: 470±40?nm band pass filter ?detection: 535±50?nm band pass filter) fluorescence channels. Flow cytometric analysis was assayed with the JC-1 Mitochondrial Membrane Potential Kit (AAT Bioquest Sunnyvale CA USA) according to the manufacturer's directions using a FACSCalibur and the results were analyzed by CellQuest software. In vitro casapse-9 activity determination Caspase-9 activity was measured by a fluorometric assay in whole-cell lysates using Ac-Leu-Glu-His-Asp-MCA substrate (Peptide International Inc. Louisville KY USA). A549 cell extracts were mixed with Ac-LEHD-MCA in ICE standard buffer (100?mM HEPES pH 7.5 10% sucrose 0.1% CHAPS 10?mM DTT 1?mM PMSF) and cleavage of the fluorogenic peptide substrate was monitored at 37?°C for 30?min by a SPECTRA max GEMINI EM (Molecular Device Sunnyvale CA USA) fluorometer with excitation at 370?nm and emission at 460?nm. This work was supported in part by a Grant-in-Aid (23591477) from the Ministry of Education Culture Sports Science and Technology of Japan. Apaf-1 apoptotic protease-activating factor 1 COX cyclooxygenase HSF-1 heat shock factor 1 MTT 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide NSAID nonsteroidal anti-inflammatory drug NSCLC non-small cell lung cancer PCR polymerase chain reaction RNAi RNA interference RT reverse transcriptase TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick and labelling Edited by G Raschellà The authors declare no conflict of interest. Tavaria M Gabriele T Kola I Anderson RL A hitchhiker's guide to the human Hsp70 family Cell Stress Chaperones 1996 1 23 28 9222585 Jaattela M Escaping cell death: survival proteins in cancer Exp Cell Res 1999 248 30 43 10094811 Aghdassi A Phillips P Dudeja V Dhaulakhandi D Sharif R Dawra R Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreas adenocarcinoma Cancer Res 2007 67 616 625 17234771 Ciocca DR Clark GM Tandon AK Fuqua SA Welch WJ McGuire WL Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer J Natl Cancer Inst 1993 85 570 574 8455204 Cornford PA Dodson AR Parsons KF Desmond AD Woolfenden A Fordham M Heat shock protein expression independently predicts clinical outcome in prostate cancer Cancer Res 2000 60 7099 7105 11156417 Vargas-Roig LM Gago FE Tello O Aznar JC Ciocca DR Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy Int J Cancer 1998 79 468 475 9761114 Igney FH Krammer PH Death and anti-death: tumor resistance to apoptosis Nat Rev Cancer 2002 2 277 288 12001989 Gabai VL Meriin AB Mosser DD Caron AW Rits S Shifrin VI Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance J Biol Chem 1997 272 18033 18037 9218432 Meriin AB Yaglom JA Gabai VL Zon L Ganiatsas S Mosser DD Protein-damaging stresses active c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by Hsp72 Mol Cell Biol 1999 19 2547 2555 10082520 Park HS Cho SG Kim GK Hwang HS Noh KT Kim MS Heat shock protein hsp72 is a negative regulator of apoptosis signal-regulating kinase 1 Mol Cell Biol 2002 22 7721 7730 12391142 Mosser DD Morimoto RI Molecular chaperones and the stress of oncogenesis Oncogene 2004 23 2907 2918 15077153 Beere HM Wolf BB Cain K Mosser DD Mahboubi A Kuwana T Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome Nat Cell Biol 2000 2 469 475 10934466 Saleh A Srinivasula SM Balkir L Robbins PD Alnemri ES Negative regulation of the Apaf-1 apoptosome by Hsp70 Nat Cell Biol 2000 2 476 483 10934467 Zermati Y Garrido C Amsellem S Fishelson S Bouscary D Valensi F Caspase activation is required for terminal erythroid differentiation J Exp Med 2001 193 247 254 11208865 Schmitt E Gehrmann M Brunet M Multhoff G Garrido C Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy J Leu Biol 2007 81 15 27 Gotoh T Terada K Oyadomari S Mori M Hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria Cell Death Differ 2004 11 390 402 14752510 Stankiewicz AR Lachapelle G Foo CZ Radicioni SM Mosser DD Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation J Biol Chem 2005 280 38729 38739 16172114 Hosokawa N Hirayoshi K Nakai A Hosokawa Y Marui N Yoshida M Flavonoids inhibit the expression of heat shock proteins Cell Struct Funct 1990 15 393 401 2085852 Yokota S Kitahara M Nagata K Benzylidene lactam compound KNK437 a novel inhibitor of acquisition of thermotolerance and heat shock protein induction in human colon carcinoma cells Cancer Res 2000 60 2942 2948 10850441 Westerheide SD Kawahara TL Orton K Morimoto RI Triptolide an inhibitor of the human heat shock response that enhances stress-induced cell death J Biol Chem 2006 281 9616 9622 16469748 Aghdassi A Phillips P Dudeja V Dhaulakhandi D Sharif R Dawra R Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma Cancer Res 2007 67 616 625 17234771 Phillips PA Dudeja V McCarroll JA Borja-Cacho D Dawra RK Grizzle WE Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70 Cancer Res 2007 67 9407 9416 17909050 Giardiello FM Offerhaus GJ DuBois RN The role of nonsteroidal anti-inflammatory drugs in colorectal cancer prevention Eur J Cancer 1995 31A 1071 1076 7576994 Thun MJ Henley SJ Patrono C Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic pharmacologic and clinical issues J Natl Cancer Inst 2002 94 252 266 11854387 Smalley WE DuBois RN Colorectal cancer and nonsteroidal anti-inflammatory drugs Adv Pharmacol 1997 39 1 20 9160111 Shiff SJ Koutsos MI Qiao L Rigas B Nonsteroidal antiinflammatory drugs inhibit the proliferation of colon adenocarcinoma cells: effects on cell cycle and apoptosis Exp Cell Res 1996 222 179 188 8549662 Klampfer L Cammenga J Wisniewski HG Nimer SD Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines Blood 1999 93 2386 2394 10090950 Shiff SJ Oiao L Tsai LL Rigas B Sulindac sulfide an aspirin-like compound inhibits proliferation causes cell cycle quiescence and induces apoptosis in HT-29 colon adenocarcinoma cells J Clin Invest 1995 96 491 503 7615821 Yao M Zhou W Sangha S Albert A Chang AJ Liu TC Effect of nonselective cyclooxygenase inhibition with low-dose ibuprofen on tumor growth angiogenesis metastasis and survival in a mouse model of colorectal cancer Clin Cancer Res 2005 11 1618 1628 15746067 Zhang L Yu J Park BH Kinzler KW Vogelstein B Role of BAX in the apoptotic response to anticancer agents Science 2000 290 989 992 11062132 Liou JY Ghelani D Yeh S Wu KK Nonsteroidal anti-inflammatory drugs induce colorectal cancer cell apoptosis by suppressing 14-3-3? Cancer Res 2007 67 3185 3191 17409426 Siddik ZH Cisplatin: mode of cytotoxic action and molecular basis of resistance Oncogene 2003 22 7265 7279 14576837 Cho HJ Kim JK Kim KD Yoon HK Cho MY Park YP Upregulation of Bcl2 is associated with cisplatin-resistance via inhibition of Bax translocation in human bladder cancer cells Cancer Lett 2006 237 56 66 16009487 Bonay M Soler P Riquet M Battesti JP Hance AJ Tazi A Expression of heat shock proteins in human lung and lung cancers A"
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" the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour and patient characteristics treatment efficacy and tolerability and quality of life better patient selection might lead to improved outcomesmethods this post hoc exploratory analysis examined the effect of prognostic factors on outcomes in the randomized double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with mcrc refractory to standard chemotherapies recourse trial patients were redivided by prognosis into two subgroups those with metastatic sites at randomisation low tumour burden and ‰¥ months from diagnosis of metastatic disease to randomisation indolent disease were included in the good prognostic characteristics gpc subgroup the remaining patients were considered to have poor prognostic characteristics ppcresults gpc patients n386 had improved outcome versus ppc patients n414 in both the trifluridinetipiracil and placebo arms gpc patients receiving trifluridinetipiracil n261 had an improved median overall survival vs months hr ci to p00001 and progression free survival vs months hr ci to p00001 than ppc patients receiving trifluridinetipiracil n273 improvements in survival were irrespective of age eastern cooperative oncology group performance status ecog ps kras mutational status and site of metastases at randomisation in the trifluridinetipiracil arm time to deterioration of ecog ps to ‰¥ and proportion of patients with ps0“ discontinuing treatment were longer for gpc than for ppc patients vs months and vs respectively low tumour burden and indolent disease were factors of good prognosis in late line mcrc with patients experiencing longer progression free survival and greater overall survivalintroductioninclusion of new therapeutic options into the current treatment landscape in metastatic colorectal cancer mcrc has led to an increased survival in the last couple of key questionswhat is already known about this subject –º the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour characteristics and patient factors as well as treatment characteristics such as tolerability efficacy and quality of life effects trifluridinetipiracil is indicated in pretreated patients with mcrc based on results of the pivotal randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial which demonstrated significantly improved overall survival os compared with placebo with a manageable safety profilewhat does this study add –º in recourse classification of patients as having good prognostic characteristics gpc defined as those with low tumour burden metastatic sites at randomisation and less aggressive disease ‰¥ months from diagnosis of first metastasis at randomisation identified a subgroup of patients with improved os and progression free survival with trifluridinetipiracil compared with patients with poor prognostic characteristics treated with trifluridinetipiracil and gpc patients treated with placebohow might this impact on clinical practice –º low tumour burden and indolent disease were shown to be factors of good prognosis in late line mcrc with these patients experiencing longer time on treatment and greater os this suggests that these patients could be candidates to receive further lines of therapy post trifluridinetipiracildecades1“ first line treatment of patients typically involves the use of vascular endothelial growth factor vegf or epidermal growth factor receptor egfr targeted agents eg bevacizumab cetuximab panitumumab to fluoropyrimidine based fluorouracil or tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesscapecitabine chemotherapy regimens depending on the presence or absence of ras mutation positive disease2 in the usa immunotherapies nivolumab±ipilimumab or pembrolizumab are also recommended for the treatment of patients with mismatch repair deficient or microsatellite instability high disease4 in the second line setting vegf targeted treatments eg aflibercept ramucirumab can also be used in combination with chemotherapy2 the optimal chemotherapeutic regimen for use beyond third line remains unclear where resistantrefractory disease and residual toxicity potentially limit the treatment options with only two possible candidates at present5the general condition and performance status of a patient are strong prognostic and predictive factors for mcrc treatment2 fitter patients are typically assigned to a more intensive treatment approach ie a combination of “ cytotoxic agents with a biological agent than less fit patients2 the choice of treatment in the metastatic setting is generally influenced by tumour characteristics tumour burden localisation and biology patient characteristics age eastern cooperative oncology group performance status ecog ps an function and comorbidities and treatment characteristics efficacy toxicity profile administration and quality of life qol effects2the proportion of patients with mcrc receiving active treatment decreases from line to line leaving more than half of patients who received an active treatment in the first line without treatment in the third line setting even in randomised clinical trials in folfiri plus cetuximab versus folfiri plus bevacizumab as first line treatment for patients with metastatic colorectal cancer only of patients reached third line6 data from the usa indicate that only of patients receiving a first line of treatment move into the second line move to the third line and only will receive a fourth line of treatment7 being unable to receive a subsequent line of treatment therefore appears to have a negative impact on the patient™s survivalis trifluridinetipiracil ftdtpi lonsurf indicated for the treatment of adult patients with mcrc who have been previously treated with or are not considered candidates for available therapies including fluoropyrimidine based oxaliplatin based and irinotecan based chemotherapies anti vegf agents and anti egfr agents for eligible patient ras wild type combination of tipiracil hydrochloride with the nucleoside metabolic inhibitor trifluridine improves its bioavailability by inhibiting its catabolism by thymidine phosphorylase8 the relatively limited non haematological toxicity of trifluridinetipiracil makes it a good option in the third line and refractory settings2 in the pivotal phase iii randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial conducted in patients with mcrc eligible for treatment in the third line and beyond treatment with trifluridinetipiracil versus placebo extended overall survival median os vs months hr p0001 and progression free survival median pfs vs months hr p000110 this effect was shown in all subgroups regardless of age ecog ps “ geographical region race and kras mutational status10 furthermore trifluridinetipiracil was well tolerated with few serious adverse events aes reported haematological toxicities were the most frequently observed aes10 also time to deterioration of ecog ps to ‰¥ was significantly improved median vs months hr p000110 with of patients treated with trifluridinetipiracil remaining at ps “ at discontinuation11 remaining at ecog ps “ is important as it could allow patients to further benefit from subsequent therapy and potentially extend their survival in recourse and of patients treated with trifluridinetipiracil remained alive at and months respectively in the refractory setting in the post hoc analysis described here we set out to explore other factors that could extend survival in the recourse population for the purposes of our exploratory analysis we defined the characteristics of good prognosis as low tumour burden metastatic sites by response evaluation criteria in solid tumors recist evaluation at randomisation and less aggressiveindolent disease ‰¥ months from diagnosis of first metastasis to randomisation which are known to be strong prognostic factors in patients with mcrc with good ecog ps12 our ultimate aim is to explore how clinicians can better predict individual treatment outcomes and support treatment selection through the continuum of carematerials and methodsstudy design and patientsthe study design and methodology of the recourse trial clinicaltrials gov number nct01607957 have been previously published10 in brief recourse was a phase iii randomised double blind placebo controlled study comparing the efficacy and safety of trifluridinetipiracil plus best supportive care with those of placebo plus best supportive care10 this study included patients with metastatic biopsy provendocumented adenocarcinoma of the colon or rectum who were previously treated with ‰¥ standard chemotherapy regimens or who had tumour progression within months of their most recent chemotherapy or who had clinically significant aes precluding readministration of standard chemotherapies patients were randomised to trifluridinetipiracil mgm2 two times a day on days “ and “ every weeks or matching placebo10 randomisation was stratified according to kras mutation status wild type vs mutant time from diagnosis of first metastasis to randomisation vs ‰¥ months and geographical region japan vs usa european union and australia10 all patients had adequate an function and were ecog ps tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0cof “ at inclusion10 the primary endpoint of the study was os and secondary endpoints included pfs objective response rate clinical benefit rate and safety10patient subgroupsin examining the effects of prognostic factors on treatment outcomes in the current analysis several subgroups of recourse patients were considered patients from recourse n800 were divided according to good prognostic characteristics gpc and poor prognostic characteristics ppc good prognosis was considered to be defined by low tumour burden metastatic sites by recist tumour evaluation at randomisation and less aggressiveindolent disease ‰¥ months from diagnosis of first metastasis to randomisation12 of the gpc subgroup n386 patients received trifluridinetipiracil and received placebo the remaining patients were included in the complementary ppc subgroup n414 of these received trifluridinetipiracil and received placeboanalysis outcomesos and pfs in the gpc subgroup were compared with those in the ppc subgroup these subgroups were then analysed according to other tumour and patient characteristics that is metastatic site at randomisation for those sites present in of the population liver lung lymph or peritoneum ecog ps vs kras mutation status wild type vs mutant and age vs ‰¥ years os and pfs with trifluridinetipiracil were compared with placebo and were analysed according to prognostic subgroups within each of the two arms finally the effect of prognostic classification of patients on ecog ps deterioration was analysed for all patients and subgroupsstatistical methodsdemographic and baseline characteristics of patients were summarised by treatment arm and subgroups using descriptive statistics n mean sd median minimum and maximum andor frequency distributions as appropriatethe differences in os pfs and time to ecog ps deterioration between trifluridinetipiracil and placebo patients or between subgroups of patients in a specific arm of treatment were assessed using the stratified log rank test stratification factors used for the randomisation from a cox proportional hazards model for each arm or each subgroup survival was summarised using kaplan meier curves and was further characterised in terms of the median with the corresponding two sided cisresultspatientsbaseline patient demographics and clinical characteristics were generally similar between gpc and ppc patients table in the trifluridinetipiracil arm slight imbalances were seen in ecog ps more gpc than ppc open accesspatients had an ecog ps of and kras status more gpc than ppc patients were kras wild type also more gpc than ppc patients had received ‰¥ prior regimens among the ppc group treated with trifluridinetipiracil of patients had ‰¥ months from diagnosis of first metastasis to randomisation but had ‰¥ metastatic sites and of patients had metastatic sites but months from diagnosis of first metastasis similar differences were observed in the placebo arm with the exception of kras status which was comparable in the gpc and ppc subgroupstreatmentamong trifluridinetipiracil treated patients those in the gpc group received more treatment cycles mean sd compared with patients in the ppc group mean sd online supplementary table s1 a higher proportion of gpc patients than ppc patients receiving trifluridinetipiracil had a dose delay vs respectively or dose reduction vs respectively which is consistent with a longer duration of treatment online supplementary table s1 however median dose intensity in the first four cycles was high ‰¥ and did not differ markedly between the groups cycle in the gpc group and the ppc group cycle and respectively cycle and respectively cycle and respectivelythe effect of good versus poor prognosis classifications on survivalsurvival curves for the gpc versus ppc subgroups are shown in figure median os was longer in the gpc subgroup than the ppc subgroup for both trifluridinetipiracil vs months hr ci to p00001 figure 1a and placebo vs months hr ci to p00001 figure 1b rates of month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively and month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively were also higher in gpc subgroups compared with ppc subgroups median pfs with trifluridinetipiracil was also longer in the gpc subgroup versus the ppc subgroup vs months hr ci to p00001 respective values for gpc versus ppc in the placebo arm were versus months hr p00699 pfs at and months in the ppc subgroup was and for trifluridinetipiracil and and for placebo respectively in the gpc subgroup these were and with trifluridinetipiracil and and with placebo respectivelyeffects of good prognostic factors on the relative efficacy of trifluridinetipiracilmedian os was prolonged with trifluridinetipiracil versus placebo in both subgroups but to a greater tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable baseline patient demographics and clinical characteristics according to prognosistrifluridinetipiracilplacebogpc subgroup n261 ppc subgroup n273 gpc subgroup n125ppc subgroup n141 females male asian other years to years ‰¥ yearsmedian age yearspatient age n gender n ethnicity n ecog ps n kras status n time since diagnosis of metastasis n number of prior regimens n number of metastatic sites n site of metastatic lesion n   primary site of disease n liver lung lymph peritoneum ‰¥ “ ‰¥ mutant wild type months ‰¥ months colon rectum defined as metastatic sites and ‰¥ months since first metastasis only those in more than of the intent to treat population are included liver lung lymph and peritoneumecog ps eastern cooperative oncology group performance status gpc good prognostic characteristics ppc poor prognostic characteristicsextent in the gpc subgroup than in the ppc subgroup figure 2a similarly median pfs was prolonged with trifluridinetipiracil versus placebo in both subgroups with the greatest magnitude of benefit observed in the gpc patients figure 2banalysis of prognostic factorsthe effect of various prognostic factors on median os and pfs is shown in table their effect on month and month os and month month and month pfs is shown in online supplementary tables and for both tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessfigure overall survival os for the good prognostic characteristics gpc and poor prognostic characteristics ppc subgroups in patients receiving a trifluridinetipiracil or b placebo ap0001 one sided bp0001 two sided ftdtpi trifluridinetipiracil mos median overall survival nr not reachedtrifluridinetipiracil and placebo the gpc subgroup had better median os and pfs than the ppc subgroup irrespective of patient age ‰¥ vs years ecog ps vs kras mutation status mutant vs wild type and liver metastases yes vs nowhen analysing the gpc subgroup the absence of liver metastasis at randomisation n153 representing of the gpc and of the intent to treat population was found to be the best factor of prognosis further information on this group of patients is available in online tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessa overall survival os b progression free survival pfs and c time to eastern cooperative oncology group figure performance status ecog ps ‰¥ with trifluridinetipiracil versus placebo in the good prognostic characteristics gpc n386 and poor prognostic characteristics ppc n414 subgroups ftdtpi trifluridinetipiracil mos median overall survivalsupplementary table s4 and online supplementary figures s1 s3 among gpc patients treated with trifluridinetipiracil median os was months longer in patients with no liver metastases compared with those with liver metastases vs months table the month os rate in gpc patients treated with trifluridinetipiracil was in those without liver metastases and in those with liver metastases corresponding month os rates in these groups were and respectively online supplementary table s2 median os was also longer in patients with no liver metastases compared with those with liver metastases in the trifluridinetipiracil ppc subgroup vs months and both the gpc and ppc subgroups of the placebo arm vs months and vs months respectively table in the group of ppc patients treated with trifluridinetipiracil the month and month os rates were and respectively in those without liver metastases compared with and respectively in those with liver metastases online supplementary table s2 for the trifluridinetipiracil and placebo arms patients with baseline ecog ps had higher median os compared with ecog ps patients in both the gpc and ppc subgroups table in the trifluridinetipiracil arm age or ‰¥ years and kras status did not seem to affect the treatment outcome table similar results were found for pfs with an effect for all trifluridinetipiracil gpc and ppc subgroups with median pfs values ranging from to months table among gpc patients treated with trifluridinetipiracil the month pfs rate was in those with no liver metastases compared with in those with liver metastases corresponding month pfs rates in the ppc group of patients treated with trifluridinetipiracil were and respectively online supplementary table s3 no such effect was observed in the placebo arm with values ranging “ months whatever the prognosis at the outset for almost all subgroups median tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0ctable the effect of various prognostic factors on median overall survival os and progression free survival pfsnumber of patientsftdtpi placebomedian survival monthshr cinumber of patientsftdtpiplacebomedian survival monthshr ciopen accessosgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgrouppfsgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroup vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs vs vs to to to to to to to to to to to to to to to to to to to to to to to to to to to to liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age‰¥ yearsn124n53n110n65kras mutantn119n64n153n71liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age ‰¥ yearsn124n53n110n65kras mutantn119n64n153n71 vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to good prognostic characteristics gpc were defined as metastatic sites at randomisation and ‰¥ months from first metastasis to randomisationftdtpi trifluridinetipiracil ppc poor prognostic characteristics ecog ps eastern cooperative oncology group performance statustabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable effects of prognostic classification of patients on eastern cooperative oncology group performance status ecog psmedian time to deterioration to ecog ps ‰¥ monthsftdtpiplaceboitt population n80011good prognosis patients n386poor prognosis patients n414ftdtpi trifluridinetipiracil itt intent to treatpfs was longer and all hrs favoured treatment with trifluridinetipiracil table effects of prognostic classification of patients on ecog psdata relative to the effect of ecog ps are presented in table the proportion of gpc patients treated with trifluridinetipiracil with an ecog ps of “ at treatment discontinuation was among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of “ at treatment discontinuation similarly among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of “ at treatment discontinuation the median time to deterioration of ecog ps to ‰¥ in patients receiving trifluridinetipiracil was months in the gpc subgroup and months in the ppc subgroup figure 2ctolerability and safetythe most common aes in patients receiving trifluridinetipiracil were nausea anaemia neutropenianeutrophil count decrease diarrhoea fatigue and reduced appetite online supplementary table s5 the most common grade ‰¥ aes experienced by patients receiving trifluridinetipiracil were haematological anaemia neutropenianeutrophil count decrease white blood cell count decrease there was no evidence of a higher incidence of aes in patients with ppc versus gpc in the group receiving trifluridinetipiracil but there was a trend towards a higher incidence of aes in placebo recipients with ppc compared with gpc online supplementary table s5discussionthe results of our analysis show that patients in the gpc subgroup consistently performed better than those in the ppc subgroup in both the trifluridinetipiracil and placebo arms within the same subgroups patients treated with trifluridinetipiracil performed better than placebo trifluridinetipiracil has consistently been shown to provide a significant survival benefit to patients with mcrc refractory to standard therapy with a well tolerated safety profile in three large scale randomised clinical trials10 “ a previous subanalysis of recourse showed that trifluridinetipiracil was more effective than placebo in patients irrespective of region age racialhr ci to to to p valueecog ps “ at treatment discontinuation ftdtpiplaceboethnic differences or kras mutation status17 in the current analysis further categorisation of patients as having good prognosis using the criteria of metastatic sites by recist tumour evaluation at randomisation and ‰¥ months from diagnosis of first metastasis to randomisation12 identified a subgroup of patients with improved os and pfs with trifluridinetipiracil compared with poorer prognosis patients ie those with ‰¥ metastatic sites and months from first metastasis pfs and os were also improved in gpc patients treated with trifluridinetipiracil compared with gpc patients who received placebopatients with gpc received more cycles of treatment than patients with ppc because progression was delayed in this group which may have contributed to the better survival outcomes the difference cannot be explained by a difference in dose intensity since this was high and similar in both the ppc and gpc subgroups of patients receiving trifluridinetipiracil in addition there was no evidence for higher toxicity in the ppc than the gpc group in fact the haematological aes occurred at a slightly higher rate in gpc patients than in ppc patients who received trifluridinetipiracil which probably reflects a longer exposure to treatment in the gpc group more patients in the gpc than in the ppc subgroup had dose delays which suggests that grade ‰¥ haematological aes were appropriately managed during treatmentit is thought that the availability of more treatment options for mcrc has contributed to an improvement in os over the last years3 indeed a retrospective study in elderly patients aged ‰¥ years a patient population more prone to comorbidities poor performance status and the development of treatment related toxicity reported a correlation between os and the number of treatment lines received18 thus maintaining the general condition and performance status of a patient throughout the continuum of care is of great importance especially beyond the second line to ensure patients remain fit with good qol5 our analysis showed that the majority of patients in the gpc subgroup discontinued treatment with an ecog ps of “ at the time of disease progression suggesting that these patients could be candidates to receive further lines of therapy post trifluridinetipiracil this is important when sequencing through the continuum of care this is in line with other tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0canalyses indicating preservation of health related qol on treatment of patients with mcrc with trifluridinetipiracil19 while the post hoc nature of this analysis limits it to an exploratory analysis the relatively large number of patients analysed make these data a good tool to estimate the expected outcomes when treating patients with refractory mcrc with trifluridinetipiracil the smaller size of some of the subgroups may limit the s that can be drawn thus preventing an evaluation of other parameters that might impact on outcomes such as lactate dehydrogenase levels the exact definition of good and poor prognostic factors12 may require further validation in a prospective cohortthe current analysis shows that compared with poor prognosis patients treated with either trifluridinetipiracil or placebo and good prognosis patients treated with placebo patients with gpcs treated with trifluridinetipiracil adequate an function ecog ps “ metastatic sites by recist tumour evaluation at randomisation and ‰¥ months from diagnosis of first metastasis have an increased survival in terms of median os and month and month survival rates treatment with trifluridinetipiracil is effective and provides the majority of patients the opportunity to maintain ecog ps and the possibility to receive further treatment options through the continuum of careauthor affiliations1vall d™hebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall d™hebron institute of oncology uvic ucc iob quironmedical oncology vall d'hebron hospital barcelona catalunya spain3medical oncology ospedale policlinico san martino istituto di ricovero e cura a carattere scientifico per l'oncologia genova liguria italy4department of medical oncology university hospital centre besançon besancon bourgogne franche comté france5kashiwa national cancer center hospital east kashiwa chiba japan6department of medical oncology dana farber cancer institute boston massachusetts usa7centre of excellence methodology and valorization of data centex mvd institut de recherches internationales servier suresnes france8global medical affairs les laboratoires servier sas suresnes île de france france9digestive oncology ku leuven university hospitals leuven leuven flanders belgiumacknowledgements the authors would like to thank andrea bothwell who wrote the first draft of this manuscript on behalf of springer healthcare communications this medical writing assistance was funded by institut de recherches internationales servier suresnes francecontributors jt and srmv contributed to the conception and design of the study all authors were involved in the acquisition analysis and interpretation of data and in writing andor revising drafts of the manuscript all authors have read and approved the final draft of the manuscript and accept responsibility for the finished article and the decision to submit the manuscript for publicationfunding the recourse study was funded by taiho oncology and taiho pharmaceutical co this analysis was funded by servier in partnership with taihocompeting interests jt has received personal fees from array biopharma astrazeneca bayer ag beigene boehringer ingelheim chugai genentech open accessgenmab as halozyme imugene limited inflection biosciences limited ipsen kura oncology eli lilly and company merck menarini merck serono merrimack pharmaceuticals merus molecular partners novartis peptomyc pfizer pharmacyclics proteodesign sl rafael pharmaceuticals f hoffmann la roche sanofi seattle genetics servier symphogen taiho pharmaceutical vcn biosciences biocartis foundation medicine haliodx sas pharmaceuticals and roche diagnostics ga has had an advisory role or received honoraria or travel grants from hoffmann la roche merck serono amgen sanofi bayer servier and bristol myers squibb afs has had an advisory role for amgen bayer celgene roche merck serono sanofi and servier and has attended a speakers™ bureau for amgen astrazeneca bayer bristol myers squibb celgene lilly merck serono roche sanofi and takeda evc has received research funding from amgen bayer boehringer ingelheim celgene ipsen lilly merck merck kga novartis roche sanofi and servier and has attended advisory boards for astellas astrazeneca bayer bristol myers squibb celgene lilly merck sharp dohme merck kgaa novartis roche and servier cb has attended advisory boards for roche servier and sanofi and has received a research grant from roche ao has received honoraria from ono bms chugai taiho eisai and amgen and has received research funding from bristol myers squibb an immediate family member of ao has been employed by celgene rjm declares no conflicts of interest lv and srmv are employees of servierpatient consent for publication not requiredethics ap
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"carcinogenesis is a process of somatic evolution previous models of stem and transient amplifying cells in epithelial proliferating units like colonic crypts showed that intermediate numbers of stem cells in a crypt should optimally prevent progression to cancer if a stem cell population is too small it is easy for a mutator mutation to drift to fixation if it is too large it is easy for selection to drive cell fitness enhancing carcinogenic mutations to fixation here we show that a multiscale microsimulation that captures both withincrypt and betweencrypt evolutionary dynamics leads to a different epithelial tissues are metapopulations of crypts we measured time to initiation of a neoplasm implemented as inactivation of both alleles of a tumor suppressor gene in our model time to initiation is dependent on the spread of mutator clones in the crypts the proportion of selectively beneficial and deleterious mutations in somatic cells is unknown and so was explored with a parameter when the majority of nonneutral mutations are deleterious the fitness of mutator clones tends to decline when crypts are maintained by few stem cells intercrypt competition tends to remove crypts with fixed mutators when there are many stem cells within a crypt there is virtually no crypt turnover but mutator clones are suppressed by withincrypt competition if the majority of nonneutral mutations are beneficial to the clone then these results are reversed and intermediatesized crypts provide the most protection against initiation these results highlight the need to understand the dynamics of turnover and the mechanisms that control homeostasis both at the level of stem cells within proliferative units and at the tissue level of competing proliferative units determining the distribution of fitness effects of somatic mutations will also be crucial to understanding the dynamics of tumor initiation and progressionk e y w o r d scancer evolution initiation metapopulation dynamics neoplastic progression simulationmajor findings competition between epithelial units such as colonic crypts tends to suppress initiation of neoplasms by suppressing mutator clones this suppression of initiation is enhanced when crypts have few stem cells and so are likely to go extinct due to stochastic fluctuations in stem cell numbers 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 evolutionary applications published by john wiley sons ltdevolutionary applications “ wileyonlinelibrarycom eva 2003 2003 0c 2003 2003 2003 2002 2003 2003introductionthe anization of a population into spatially distinct subpopulations can have a dramatic effect on the evolution of that metapopulation hanski gaggiotti this has implications for both the evolution of anisms and for the effect of tissue architecture on somatic evolution and tissue health in multicellular anisms epithelia are typically divided into subpopulations of tissue stem cells along with the transient amplifying cells and differentiated cells that they produce these subpopulations go by different names in different tissues such as crypts in the intestine or more generally epithelial proliferative units cairns first recognized that the division of stem cells into subpopulations such as crypts acts as a tumor suppressor cairns a mutant stem cell with a reproductive or survival advantage may take over a crypt but is generally constrained from expanding beyond that subpopulation unless it breaches the crypt via a process known as œcrypt fission which tends to duplicate the mutant crypt cell population however by establishing a population size barrier the mutant clone has to overcome the subpopulation structure of the tissue limits the probability that that clone will acquire further carcinogenic mutations yet clones of mutant stem cells can be observed at scales spanning many crypt diameters especially in compromised tissues such as ulcerative colitis and barrett's esophagus maley salk a fundamental question is therefore how the cryptlevel metapopulation dynamics affect the accumulation of somatic mutations during carcinogenesishere we explore the evolutionary dynamics of mutant stem populations that lead to tumor initiation that is the breach of the crypt barrier allowing clonal expansions of crypts across a tissue as well as of mutant stem cells within and out of a crypt while there may be multiple pathways to tumor initiation it has been shown that the inactivation of a single tumor suppressor gene tsg such as the adenomatous polyposis coli apc gene in colon is sufficient to abrogate crypt homeostasis leading to the formation of aberrant crypts and nascent adenomas humphries wright using an agentbased microsimulation model for both stem cell turnover within a crypt and for the cryptpopulation tissuelevel dynamics we study the role of pretumor evolution in tumor initiation this exploration allows for the selection of mutant crypts across the tissue prior to the inactivation of the tumor suppressor gene”a form of premalignant field cancerization”while the stem cell in which the tumor suppressor is inactivated can proliferate beyond the limit of a single crypt due to crypt bifurcationthe evolution of somatic cells is a complex multiscale process depending on the nature of somatic mutations which may either increase or decrease cell fitness stem cell divisions and differentiation or apoptosis as well as subpopulation eg crypt division and extinction rates there is considerable evidence that carcinogenesis involves both an increase in the rate of epigenetic lesions bielas loeb rubin true loeb breivik ji king weisenberger et al and expansions of clones with a relative fitness advantage over their competitor cells cannataro gaffney townsend maley pepper findlay kassen spencer maley vermeulen williams however it continues to be unclear whether the mutator phenotype is a preinitiation phenomenon in carcinogenesis or is more likely to occur during tumor progression in barrett's esophagus another cryptstructured precancer we found evidence that genomic instability precedes genome doubling and transformation martinez the frequency of deleterious versus beneficial mutations in somatic cells is also unknown though the large number of genes in metazoans devoted to differentiation apoptosis and cell cycle control suggests that the frequency of deleterious mutations may be lower in somatic evolution than anismal evolution rajagopalan nowak vogelstein lengauer recent analysis of somatic mutations in cancer found no evidence of purifying selection except in a few essential genes and strong evidence of positive selection with large selective effects williams suggesting that beneficial mutations are more common than deleterious mutations in somatic evolution martincorena although a definitive answer to these questions can only come from further experimental data a theoretical exploration that recognizes the roles of metapopulation dynamics the mutator phenotype and the proportion of deleterious to advantageous mutations in the process of tumor initiation is called for such an exploration will help the identification of factors that drive the tumor initiation processour model integrates previous efforts to characterize the stem cell dynamics within a crypt cannataro mckinley st mary cannataro mckinley st mary frank iwasa nowak komarova komarova cheng loeffler birke winton potten meineke potten loeffler michor frank may iwasa nowak nowak et al pepper sprouffske maley with models of the dynamics of crypt populations cannataro et al chao eck brash maley luebeck kostadinov maley kuhner loeffler bratke paulus li potten totafurno bjerknes cheng mathematical studies of the stem cell population in the crypt niche suggest that epigenetic alterations that increase the rate of genetic lesions mutator mutations and reduce the fitness of stem cells will tend to drift to fixation if the stem cell population is small whereas carcinogenic mutations that increase the proliferation or survival of a stem cell will tend to spread if the stem cell population is large cannataro komarova michor assuming that most nonneutral somatic mutations are deleterious the accumulation of deleterious mutations may lead to senescence of the intestine over time cannataro however competition between crypts of different fitnesses may significantly change the dynamics of the establishment of a mutator clone through a metapopulation dynamic our in silico experiments suggest that there may have been selection at the level of the anism to minimize the number of stem cells within each subpopulation of birtwell 0c 2003 2002 2003 2003its structured epithelium so as to reduce the probability of tumor suppressor gene inactivation and the initiation of carcinogenesisproliferation rate decreased stem cell loss and caused the mutator phenotypethe following equations and assumptions govern the model 2003 2003methodswe implemented a multiscale model of epithelial tissue architecture with stem cells subdivided into crypts under homeostatic control we examined the time required until the two alleles of a tumor suppressor gene tsg were inactivated in at least one stem cell to represent tumor initiation the model was run at least times for every parameter setting crypts were arranged in a flat hexagonal tissue similar to that observed in colon and contained a population of stem cells as well as an implicitly modeled transient amplifying compartment stem cells divided both symmetrically and asymmetrically symmetric division resulted in two daughter stem cells each having the opportunity during the division event synthesis to acquire a mutation asymmetric division did not result in any new stem cells but did provide an opportunity for stem cell mutation stem cell loss due to cell death or differentiation and stem cell gain due to division events were modeled as a stochastic birth“death process with parameters that were functions of the stem cell fitness and of homeostatic feedback effects in response to deviations of the crypt cell population from its normal target level equations and a flow chart of the model algorithm is shown in figure s1homeostasis operated at two spatial scales within a crypt if the stem cell population dropped below the target level stem cell division rates increased by a parameterized amount equation if the population rose above the target level stem cell loss rates increased equation the level of homeostatic feedback was proportional to the degree of deviation away from the target equilibrium level equations and we also introduced a mechanism for homeostasis on the hexagonal lattice of crypts if all the stem cells in a crypt died the inhibition on stem cell population growth was released from the neighboring crypts when the stem cell population of a neighbor reached twice the equilibrium level we modeled crypt bifurcation by allocating half of its stem cells to a new crypt in the location of the dead neighborwe included both beneficial mutations that increased the division probability or the survival probability of stem cells as well as deleterious mutations that decreased them these can accumulate indefinitely and affect fitness multiplicatively equation we also implemented a genetic instability mutation that increased the clone's mutation rate 100fold bielas herr kennedy knowels schultz preston ji king the frequency of each mutation type those that changed the cell's fitness the proportion of nonneutral mutations that were deleterious as well as the rate of tsg inactivation was set by parameters each mutation affected proliferation survival or mutation rate parameters by a constant factor half of the deleterious mutations decreased stem cell proliferation and half decreased their survival increased cell loss for the beneficial mutations increased the cell's 2003 2003equationsequation time to stem cell losslet t be a random exponential deviate with distribution function fr t and rate parameter r the time to cell loss due to apoptosis or differentiation is the minimum of the time to cell loss due to background cell death or differentiation and the time to cell loss due to crypt feedbacktcell_loss min 01t1 ˆ¼ fbackground_loss t2 ˆ¼ ffeedback_loss 01 equation homeostatic crypt feedback by differentiationwhen the stem cell population within a crypt expands beyond the homeostatic target level kcrypt\\_size the crypt provides homeostatic feedback via a change in the rate of stem cell loss with a rate parameter equal to the base stem cell loss rate multiplied by the crypt feedback multiplier the crypt feedback rate multiplier is used to calculate the time to stem cell loss due to crypt homeostatic feedback the crypt feedback multiplier is equal to raised to the nth power where n is the excess number of stem cells above the crypt size divided by the kcrypt\\_deviation parameter here kcrypt\\_deviation and kcrypt\\_size is a parameter that we varied across experimentsrfeedback\\_cell_loss rbase_cell_loss2max0ncellsˆ’kcrypt_sizeˆ•kcrypt_deviationequation homeostatic crypt feedback by proliferationwhen the stem cell population of a crypt drops below kcrypt\\_size the division rate of the remaining stem cells is increased by a factor that depends on the difference between the current number of stem cells ncellsand kcrypt\\_sizerfeedback_division rbase_division2max0kcrypt_sizeˆ’ncellsˆ•kcrypt_deviationequation fitness mutation effectskfitness is a constant factor representing the effect of a single beneficial mutation on fitness as a first approximation we assume that there are many possible mutations that increase and decrease the fitness of a somatic clone by approximately the same amount and so the effect of n beneficial mutations nbeneficial on stem cell fitness is the constant fitness effect raised to the nth power the effect of n deleterious mutations ndeleterious of small effect is just the inverse of kfitness raised to the nth power there is a separate mfitness calculated for the division probability and the survival probability of a cell because beneficial and deleterious mutations may affect either of those probabilitiesmfitness 01kfitness 01nbeneficial 03 kfitness 03ndeleteriousbirtwell 0c 2003 2003 2003 2002f i g u r e 2003plots of cumulative hazard functions using the kaplan“meier estimator the tissue was x crypts with stem cells per crypt in panels a through d each colored line represents the function for a specific proportion of deleterious mutations a baseline experiment with default parameter values table b mutation rate reduced to 01x of baseline c mutator phenotype reduced to 01x of baseline d each colored line represents a different number of cells per crypt all proportions of deleterious mutations were included 2003 2003assumptionscrypts consist of stem cells and of transient amplifying cellscrypt density is fixed that is the tissue contains a fixed number of crypts arranged on a hexagonal griddrops below the target level the division rate of each stem cell in the crypt is increased when the number of stem cells grows above the target level the cell loss rate of each stem cell in the crypt is increasedcrypts divide to fill vacant slots left by adjacent crypts that have the number of cells in a crypt transient amplifying compartment gone extinct due to loss of the constituent stem cellsis fixedcrypts attempt to maintain a stable population of stem cells through homeostatic feedback when the number of stem cells the extinction of an adjacent crypt suppresses the homeostatic apoptotic signals allowing the stem cell populations in neighboring crypts to expand once that extinct crypt is replaced the normal birtwell 0cta b l e 2003baseline simulation parameterssimulationmaximum simulation duration in daysstem celldivision rate rbase\\_divisionratio of asymmetric divisions to symmetric divisionsstem cell loss rate rbase\\_cell\\_lossmutation rate per stem cell divisionmutation rate maximumtumor suppressor gene mutation ratetumor suppressor gene mutation rate maximumnumber of transient amplifying cells associated with each stem cellcell division minimum time in dayscell loss minimum time in dayscrypttarget equilibrium level of number of stem cells in a crypt kcrypt\\_sizestandard deviation from equilibrium level the crypt uses this value to determine its level of effect on the cell loss and division rates of the stem cells kcrypt\\_deviationbifurcation threshold factor below which a crypt will not bifurcatecrypt cell loss effect multipliercrypt division effect multipliermultiplier to the division effect for each dead neighbor cryptcanceruncontrolled cell proliferation threshold if a crypt has this threshold times the equilibrium number of stem cells it is considered to be experiencing uncontrolled stem cellnumber of tumor suppressor gene hits that mean cancermutation 2003 2002 2003 2003 years x “ x “ x “ also tested x “ x “default varied from “percent of nonneutral mutations that are deleteriousthe factor affecting the loss rate of the stem cell from a beneficial mutation ˆ•kfitnessthe factor affecting the division rate of the stem cell from a beneficial mutation kfitnessthe factor affecting the cell loss rate of the stem cell from a deleterious mutation kfitnessthe factor affecting the division rate of the stem cell from a deleterious mutation ˆ•kfitnessthe factor affecting the mutation rate of the stem cell from a mutator mutation“ also tested homeostatic controls on stem cell numbers of neighboring crypts are restoredcrypt division is triggered by an expansion of the stem cell population of a crypt to twice its homeostatic level as hypothesized by garcia park novelli and wright as long as there is an empty slot adjacent to the enlarged crypta stochastic birth“death process governs the scheduling of division and cell loss eventsfitness mutations affect in a multiplicative fashion the rate parameters of the birth“death processthere is a single mutator phenotype that requires only a single mutator mutation additional mutator mutations have no effect on the mutation ratethe loss of the first allele of the tsg has no effect on stem cell fitness 2003 2003results 2003 2003tsg inactivation depends on the emergence of a mutatorat baseline for comparison our tissue was a 5x5 hexagonal lattice of crypts each crypt having stem cells stem cell loss and symmetric division rates were balanced mutations were acquired stochastically with probabilities defined by proportions starting with deleterious mutations beneficial mutations and mutator mutations and ranging in increments to deleterious beneficial and mutator beneficial versus mutator the incidence of tsg inactivation decreased as the proportion of deleterious mutations increased figure 1a table birtwell 0c 2003 2003 2003 2002reducing the base mutation rate to of baseline we observed a marked decrease in tsg inactivation figure 1b as expected similarly reducing the effect of the mutator mutation to 10x the baseline mutation rate instead of 100x produced a significant decrease in tsg inactivation figure 1c we found that the vast majority of tsg inactivation occurred in stem cells that had previously acquired the mutator phenotype figure s2 this assumes that the mutator phenotype can be caused by a single mutation that is otherwise neutral eg overexpression of dna polymerase beta canitrot or a dominantnegative mutation in p53 de vries though this assumption is easily relaxed 2003 2003proportion of deleterious mutations negatively correlates with tsg inactivationnot surprisingly we found that the proportion of deleterious mutations and the incidence of tsg inactivation were negatively correlated figure cell divisions per time remained roughly constant across all proportions of mutations however as the proportion of deleterious mutations decreased the cost of being a mutator also decreased because it accumulated less mutational burden of deterious mutations this resulted in an increased emergence of crypts with fixed mutator stem cell populations conversely across all experiments we observed progressively less tsg inactivation as the proportion of deleterious mutations approached our maximum of turnover levels we observed a reduction in the average number of stem cells per crypt figure s3 the implemented homeostatic control was unable to maintain the target stem cell population size in the face of high turnover rates essentially there is a lag between depletion of the stem cell pool due to cell death and differentiation and replenishment provided by an increase in stem cell division rates with higher levels of cell loss the simulated crypts spend more time further below the target homeostatic number of stem cells as a result there were fewer total stem cells in the simulation and therefore fewer mutations per time allowing less chance for mutator acquisition and tsg inactivation this may or may not be realistic second as the turnover level increased above our baseline the number of mutator crypts present in the tissue at any given time decreased figure since increased turnover should lead to increased opportunities for mutator mutations to arise the decline in mutator crypts was a surprise however the loss of mutator crypts is due to intercrypt competition as described below increased turnover led to increased stochastic fluctuations in stem cell numbers and thereby increased crypt extinction events the reduction in stem cell numbers and mutator crypts combined to produce a reduction in the overall incidence of tsg inactivation as turnover rates increased above baseline if in reality homeostatic control of stem cell numbers prevents increased crypt extinctions with increased stem cell turnover this result would likely not hold however all things being equal increased cell turnover would be expected to increase stem cell number fluctuations 2003 2003increased stem cell turnover initially increased and then decreased tsg inactivationwe modulated stem cell turnover by varying cell loss and symmetric division rates in unison at lower turnover levels we found that increased cell turnover increased tsg inactivation however at turnover levels 2x and 5x our baseline level the incidence of tsg inactivation declined figure due to two factors first at higher 2003 2003the number of stem cells per crypt had a varying effect on tsg inactivationin general fewer stem cells per crypt reduced the rate of tsg inactivation even though the total number of stem cells in the tissue was held constant figure 1d however there is a tradeoff between tsg inactivation and tissue death at very low stem cells per crypt with only one stem cell per crypt there is no opportunity for homeostatic signals within a crypt to compensate for stem cell loss in f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed each bar corresponds to a specific proportion of deleterious mutations the proportion of mutator crypts correlates with the risk of tumor initiation as seen in figure birtwell 0cbase cell loss divison rate base cell loss divison rate 2003 2002 2003 2003base cell loss divison rate base cell loss divison rate f i g u r e 2003the effect of changes in stem cell turnover plots of cumulative hazard functions using the kaplan“meier estimator where each colored line represents the function for a specific proportion of deleterious mutations the baseline division and stem cell loss rates were as seen in figure initially turnover correlated positively with the risk of tumor initiation however at higher turnover rates the risk of tumor initiation decreased due to a reduction in the overall number of living stem cells and decreased incidence of mutator cryptsour model tissues with one stem cell per crypt were mostly unviable and died out before tsg inactivation or the predetermined simulation end timeat and cells per crypt we observed a reduction in the incidence of tsg inactivation figure 1d at all but the lowest proportions of deleterious mutations as was predicted by models of the crypt stem cell niche of single crypts komarova michor figure s4 as in other experiments along with a reduction in the incidence of tsg inactivation the frequency of fixed mutator crypts was reduced as well this shows that selection against mutator cells increases as the number of stem cells increases above some threshold in our model as long as the majority of nonneutral mutations are deleterious supporting the s by michor and komarova komarova michor et al when the stem cell populations are large it is very unlikely that a crypt will go extinct and so there is no intercrypt competition in this case the metapopulation dynamics are reduced to the single crypt dynamicsthe increased risk of tsg inactivation associated with increased stem cells per crypt appeared to plateau after approximately stem cells per crypt the total number of cells in the tissue remained constant as did the total stem cell divisions per time figure s5 the average number of mutations per time increased through stem cells per crypt but then reached a temporary plateau figure there was no statistically significant difference between the average number of mutations per time in the and stem cells per crypt cases as the number of stem cells per crypt increased beyond the average mutations per time decreased except when the proportion of deleterious mutations was the incidence of mutator crypts followed a similar trend figure birtwell 0c 2003 2003 2003 2002f i g u r e 2003this graph represents the proportion of crypts at the end of the run that contained a population of stem cells with the mutator mutation fixed as a function of turnover rate each bar corresponds to a specific proportion of deleterious mutations as turnover increased mutator crypts became more rare at higher proportions of deleterious mutationsf i g u r e 2003number of mutations per unit time as a function of cells per crypt and proportion of deleterious mutations where each bar represents a specific proportion of deleterious mutations at most proportions of deleterious mutations mutations per time peaked around stem cells per crypt at deleterious mutations mutations per time reached a minimum at cells per crypt and increased as the cells per crypt grew past we have chosen to explore the case where the total number of stem cells is kept constant assuming that a certain number of selfrenewing tissue stem cells might be required to maintain an epithelial tissue an alternative view is that a fixed number of epithelial units like the crypts might be required to maintain a tissue and that the number of stem cells per crypt could vary by changing the number of differentiated cells produced by each stem cell in this case the risk of tsg inactivation continues to increase with increasing number of stem cells per crypt figure s4 because we held the number of crypts constant but changed the number of cells per crypt in this case the total number of cells in this simulation also changed leading to a large evolving population size of stem cells and thus an increased chance for at least one cell to inactivate both alleles of the tsg 2003 2003partitioning the tissue into crypts imposed a metapopulation dynamicfitness levels measured by the difference between division and loss rates were greater in tissues with smaller crypts even as the total number of stem cells remained constant figure 6a counts of crypt births and crypt life span measurements showed that there was more crypt turnover in smaller crypts figures s6 and s7 this crypt turnover provided an opportunity for fitter phenotypes to spread more easily across the tissue crypt fitness peaked at cells per crypt where there was the most crypt turnover and bottomed out at and cells per crypt after cells per crypt we observed no crypt death and therefore no turnover crypt fitness began to increase again at and cells per crypt through natural selection of stem cells within larger crypts however fitness levels did not increase to the levels seen at cells per cryptthe metapopulation dynamic was observed in the clonal expansion of mutator crypts across the tissue we considered two crypts to have œmutator phenotype agreement if they both have a fixed mutator mutation or neither has a fixed mutator we calculated mutator crypt agreement across spatial distance and found that overall agreement decreased as the cells per crypt increased figure 6b further at and cells per crypt closer crypts had increased mutator agreement suggesting clonal expansion of mutator crypts conversely at cells per crypt and above we found that spatial distance correlated less well with mutator agreement indicating that birtwell 0c 2003 2002 2003 2003f i g u r e 2003a crypt fitness measured by the difference between division and cell loss rates across cells per crypt and proportion of deleterious mutations the total number of stem cells remained constant across these experiments the crypt turnover at and cells per crypt allowed fit clones to spread across the tissue resulting in increased overall fitness b mutator agreement by distance class two crypts were in œmutator agreement if they both had fixed mutator mutations or neither did overall agreement was higher in the smaller crypts suggesting that mutator clones were able to spread across the tissuewhen there was less crypt turnover mutator crypts arose de novo rather than through cryptlevel clonal expansion 2003 2003discussionin the colon the development of adenomatous polyps frequently involves the inactivation of the apc gatekeeper gene a member of the wntsignaling pathway which represses proliferation and facilitates orderly cell differentiation in the luminal part of the crypts barker goss groden as long as this gatekeeper gene is active mutant stem cell progeny with neoplastic potential is likely eliminated from the crypt and clonal expansion thus averted however when the gatekeeper gene is inactivated the brakes on mutant stem proliferation are removed and mutant cell progeny may undergo focal clonal expansion therefore we asked the question what are the factors that determine the risk of a tsggatekeeper inactivation leading to tumor initiation in a compartmentalized tissue a metapopulationour microsimulations indicate that the base mutation rate figure 1b and the increase in that rate for a mutator clone figure 1c are the main driving forces behind tsg inactivation and thus the initiation of carcinogenesis the proportion of deleterious mutations is important for its effect on selection of the mutator clone we find that if most mutations are assumed to be deleterious then a mutator clone quickly accumulates a large genetic load of deleterious mutations and tends to be driven to extinction in competition with nonmutator cells however if mutations are more likely to be beneficial then a mutator clone can spread more easily which leads to the early inactivation of both alleles of the tumor suppressor gene figure 1a similarly increasing turnover of the stem cells effectively increases the mutation rate and consequently the rate of initiation however when turnover is very high the homeostatic feedback signals cannot maintain the target number of stem cells and so the total stem cell population of the epithelium decreases which in turn decreases the chances for initiation figure since the rate of tsg inactivation is trivially relat
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"elucidate the molecular mechanism underlying the involvement of abnormal DNA methylation in the development of glioma and identify potential newtargets for glioma therapyMethods The GSE79122 chip achieved from the Gene Expression Omnibus GEOdatabase containing glioma samples and normal samples was analyzed Methylationspecific polymerase chain reaction MSPCR or MSP reverse transcriptionPCR andWestern blot analysis were used to confirm the methylation level and expression level ofthe interleukin receptorassociated kinase IRAK3 gene in glioma cells glioma samplesand the corresponding normal samples In vitro the proliferation apoptosis rate migrationand invasion abilities of glioma cells were detected by Cell Counting Kit8 assay Transwellassay enzymelinked immunosorbent assay and flow cytometry respectively Besides thexenograft assay of nude mice was used to confirm the effect of the IRAK3 on glioma in vivoResults Microarray analysis showed that the IRAK3 was one of the most hypermethylated genesin glioma and the related mitogenactivated protein kinase MAPK signaling pathway wasactivated More experiments supported the higher methylation level and lower expression levelof the IRAK3 in glioma tissues and cell lines The viability migration and invasion ability ofglioma cells significantly reduced and the apoptosis rate increased with the overexpression anddemethylation of the IRAK3 in vitro Besides treatment with the MAPK signaling pathwayinhibitor PD325901 alone or the overexpression or demethylation of the IRAK3 had a similareffect as the overexpression or demethylation of the IRAK3 alone in glioma cells In vivoxenotransplantation experiments in nude mice confirmed that the overexpression and demethylation of the IRAK3 and suppression of the MAPK signaling pathway inhibited the development ofgliomaConclusion IRAK3 inhibited the development of glioma progression through the MAPKsignaling pathwayKeywords glioma IRAK3 MAPK signaling pathway methylationIntroductionGlioma also known as glioblastoma is one of the most common primary malignant braintumors The average incidence rate of glioma is in individuals1 Despiteimprovements in neurosurgery and radiotherapychemotherapy most patients die within months of diagnosis1 and less than patients survive for years or even more2Recently the molecular mechanisms of glioma have gained increasing attention so as tofind some better methods to defeat this disease Previous studies evaluated that longtermsurvivors of glioma often displayed some favorable molecular characteristics such as thesubmit your manuscript wwwdovepresscomDovePresshttp102147CMARS252772Cancer Management and Research “ Wu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphpand incorporate the Creative Commons Attribution “ Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the workyou 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 Forpermission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWu et alDovepresshypermethylation of O6methylguanine DNA methyltransferase MGMT promoter3 which is known as a meaningfulpredictive biomarker for the favorable prognosis of the chemotherapeutic drugs4 Therefore this study proposed that epigenetic regulation might play a key role in the development ofglioma which deserves further research for understanding thiscancerDNA methylation isan epigenetic modificationinvolved in many biological processes especially geneexpression regulation5 The DNA methylation patterns ofnormal cells are controlled well by a balance betweenmethylation and demethylation However this balance isalways disturbed in cancer cells through ectopic deficientor excessive methylation leading to abnormal biologicalactivities6 Hypermethylation of CpG islands on specificpromoters inhibiting the transcription of downstreamtumor suppressor genes has been discovered in manycancers which may provide clinicians a new strategy forpatients with cancer7 For instance the promoter region ofSEPT9 is hypermethylated in colorectal cancer Hence theSEPT9 gene methylation assay might become a potentialoption for the early detection and screening of colorectalcancer8 CpG islands also display aberrant hypermethylation at a large number of loci and define the subgroup ofglioma910 However the underlying molecular events ofDNA methylation and glioma development remain poorlyunderstoodRecently technical advances in genomewide expression analysis have enabled an improved understanding ofthe diagnosis and prognosis of many types of tumors11Genomewide DNA methylation analysis allows comprehensive DNA methylation profiling of the whole genomehelping in the effective identification of novel genes thathave a potential value in clinical practice12 Previous studies have reported many specific methylation signaturegenes in differenttypes of cancers such as thyroidcancer13 lung squamous cell carcinoma14 hepatocellularcarcinoma15 prostate cancer16 and so on using DNAmethylation analysisThis study aimed to examine the genomewide DNAmethylation profiling of glioma tissuesrevealing thehypermethylation of several genes in glioma Then oneof these hypermethylated genes IRAK3 was selected toconductSubsequentlya relationship between IRAK3 and MAPK signaling pathway was demonstrated by using DNA methyltransferaseinhibitor overexpression of IRAK3 and MAPK signalingpathway inhibitor which can disrupt the development ofcomprehensiveaanalysisgliomas in vitro and in vivo The findings might providesome clues for the regulatory role of DNA methylationand the underlying application of targeted treatment ingliomaMethodsTissue SamplesGlioma tissues and adjacent normal tissues were collectedfrom patients with primary glioma n admitted to theZhangye People™s Hospital Affiliated to Hexi UniversitySample collection and use was performed according to theapproval of the ethics committee of the Affiliated Hospitalof Shandong University Written informed consent wasprovided by every patient with glioma All samples werefrozen in liquid nitrogen and stored at “°C All humanspecimens were obtained with the approval by theInstitutional Ethics Committee of Zhangye People™sHospital Affiliated to Hexi UniversityCell CultureHuman gliocyte cell line HEB and glioma cell linesSHG44 U251 GOS3 HS683 and SF539 wereobtained from Bena Culture Collection Beijing ChinaHEB U251 HS683 and SF539 cells were grown in highsugar Dulbecco™s modified Eagle™s medium DMEMwith fetal bovine serum FBS SHG44 cells weregrown in RPMI1640 medium containing NaHCO3 gL glucose gL and sodium pyruvate gLwith FBS The GOS3 cells were grown with minimum essential medium with Earle™s Balanced Saltswith FBS and mML glucose All cells were cultured at °C under a humidified atmosphere with CO2Cell TransfectionpcDNA31“interleukin receptor“associated kinase pcDNA31IRAK3 was obtained from GenePharma ShanghaiChina and 5azadC 5aza2ʹdeoxycytidine 5azadCand signaling pathway inhibitor PD325901 were obtainedfrom SigmaAldrich MO USA U251 cells were seeded insixwell plates at — cellswell and cultured at °C andthe cell confluence reached “ CO2 untilTransfections were executed using Lipofectamine Invitrogen CA USA following the manufacturer™s protocols The medium was changed with the complete mediumafter h of transfectionsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressTable qRTPCR PrimersWu et alAccession NumberSequence 5ʹ3ʹForwardReverseForwardReverseForwardReverseForwardReverseACCCAAACTAACTGATTTTGCCAAGAGAAATTCCGAGGGCAGGCGACCTCCCATGGCAATTTTAACAGAGACAGGCATGGGAAGCCATCTCGACCAGTCCGTCTAGTTGGTCTGTCTCCGCTAAATACGGACTGCAGCCCTGAGGTCAATGAAGGGGTCGTGeneIRAK3MEK1CfosNM_007199NM_002755NM_005252GAPDHNM_008084GenomeWide DNA MethylationAnalysisFor DNA methylation profiling Infinium HumanMethylation450K BeadChip illuminaga_rnaseqv2100 was obtainedfrom the Gene Expression Omnibus GEO database TheDNA methylation data of chip number GSE79122 whichcontained glioma tissues glioblastomas diffuseastrocytomas and anaplastic astrocytomas and controlbrain tissues were analyzed The Infinium MethylationAbbiotec CA USA and Illumina BeadStudio softwareGenetech Biotech Taipei Taiwan were used to measurethe methylation profiles of modified DNA and loci CpGThe methylated signal intensity at particular CpG loci and450K BeadChip assay were presented as β values and percentage respectivelyQuantitative RTPCRTotal cellular RNA was extracted using PureLink Invitrogenfollowing the manufacturer™s protocol RT was performed on µg total cellular RNA using a HighCapacity cDNA ReverseTranscription Kit with RNase Inhibitor Applied Biosystemspurchased from Thermo Fisher Scientific MA USASubsequently quantitative reverse transcriptionpolymerasechain reaction RTPCR was performed using ArcturusParadise Plus qRTPCR Kit Applied Biosystems ThermoFisher Scientific Comparative expression values were calculated by the “ΔΔCt method GAPDH was used for internalreference All primer sequences involved are listed in Table converted into uracil without changing the state of methylated cytosine The IRAK3 methylation level in glioma wasidentified using methylationspecific PCR MSP The MSPprimers are listed in Table EnzymeLinked Immunosorbent AssayThe human interleukin IL6 enzymelinked immunosorbent assay kit Sangon Biotech Shanghai China was usedto test the IL6 level in the glioma cell culture medium Theglioma cell culture medium was centrifuged at rpm for min to remove cells and polymers The supernatant fluidstored at “°C was preserved in the supernatant fluid at “°C A normal glioma cell culture medium was used as thecontrolWestern Blot AnalysisLysis buffer RIPA Thermo Fisher Scientific and NPERThermo Fisher Scientific were used to extract proteinsfrom glioma cells and tissues respectively Then μg totalprotein per sample was separated using SDSPAGE andtransferred to polyvinylidene fluoride membranes ThermoFisher Scientific The membranes were probed with primaryantiIRAK3 antibody ab8116 Abcam MA USA antiMEK1 phospho S298 antibody [EPR3338] ab96379 antiERK1 ERK2 antibody [ERK7D8] ab54230 anticFosphospho T232 antibody ab17933 and antiGAPDH antibody [6C5] ab8245 Abcam as control The number ofTable MethylationSpecific PrimersDNA Methylation AssayGenomic DNA in glioma tissues and cells was extracted andtreated with bisulfite using CpGenome DNA ModificationKit Chemicon CA USA following the manufacturer™sprotocol All unmethylated cytosine residues in DNA wereGeneIRAK3 forwardIRAK3 reverseIRAK3 forwardIRAK3 reverseSequence 5ʹ3ʹ5ʹTCGGGATAGTGGTTAATATTTC3ʹ5ʹTTTTTTTCGTTTTTCGTAAAA3ʹ5ʹ AGTTTGGGATAGTGGTTAATATTTT 3ʹ5ʹ TTTTTTTCATTTTTCATAAAAAAA 3ʹCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Genomewide methylation data were obtained from the GEO database for available glioma9 adjacent mucosa A Density of methylated DNA intensity byeach sample B Type I and Type II assays showed variant β value distributions The differences between probe types were regulated by normalization procedures whichshowed that represented unmethylated sites while represented fully methylated sites C Multidimensional scaling plot showing differential clustering of control versustumor tissues D Dendrogram produced for probes in normal and tumor tissues E Heatmap of top differentially methylated imprinted CpG sitesAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Distribution of top differentially methylated imprinted CpG sites A Distribution of top differentially methylated imprinted CpG sites according toCpG islands island sea shelf and shore B Distribution of top differentially methylated imprinted CpG sites according to the position relative to genes 1stexon ² UTRs or ² UTRs body IGR TSS1500 and TSS200 C Combined genetic and epigenetic annotation information revealed the distribution of the top differentially methylated imprinted CpG sitesbinding proteins was measured using AlphaEaseFC softwareGenetic Technologies FL USACell Counting Kit8 AssayU251 cells were seeded in 96well plates and allowed toadhere for “ h at °C Then these cells were transfectedwith pcDNA31IRAK3 and treated with 5azadC orPD325901 After “ h Cell Counting Kit8 DojindoKumamoto Japan was used to test the cell viability at respective time points To summarize mL of the triazoliumsubstrate was added to each well and coincubated with cellsat °C for h The absorbance was measured at nm andCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure DNA methylation analysis of tumor tissues and normal tissues and analysis of methylation degree for seven CpG sites of IRAK3 A The expression of the top candidate genes was analyzed IRAK3 was hypermethylated significantly in tumor tissues compared with normal tissues B The number of IRAK3“methylated CpG islands ineach isosite C“I Seven CpG sites for IRAK3 which are presented in the boxplot displayed a decreased methylation in the tumor group The boxplot for cg Ccg D cg E cg F cg G cg H and cg IAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related signaling pathway A The top signaling pathways with the highest and lowest correlation with glioma B The top signalingpathway“related genes enriched in the glioma the MAPK signaling pathway was highly expressed C The MAPK signaling pathway was activated in glioma D The status ofthe top enriched signaling pathways in gliomaAbbreviation MG malignant gliomaCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressthe results were normalized to untreated cells at respectivetime pointsCell Migration and Invasion AssayBoth cell migration and invasion were performed using theTranswell assay For cell migration assay — U251cells were seeded in a serumfree medium in the upperchamber which contained a polyethylene terephthalatemembrane with mm in diameter and μm in poresize The bottom chamber was prepared with FBS asa chemoattractant After incubating at °C for hnonmigrated cells were scraped from the upper surfaceof the membrane with a cotton swab and the cells migrating to the bottom chamber were fixed with paraformaldehyde and stained with crystal violet Finally the stainedcells were counted under a microscope at — magnification in five randomly selected fields for quantificationinvasion assay — U251 cells weresuspended in mL of serumfree DMEM and thentreated using the same procedure as for the migrationassay following the manufacturer™s protocol but the chambers mm BD Biosciences San Jose CA USA wereplated with BD BioCoat MatrigelFor cellFlow CytometryEach treatment of U251 cells was washed with phosphatebuffered saline and resuspended in μL of AnnexinV binding buffer After staining with Alexa Fluor Annexin V and 7AAD Viability Staining Solution for min in the dark at room temperature these cells were analyzed using multicolor flow cytometer Data were analyzedusing Kaluza softwarethe mice were keptXenograft StudiesMale BALBc nude mice weeks were maintained underpathogenfree conditions Allina temperaturecontrolled airconditioned conventional animal house with a h light“dark cycle and given free accessto food and water Besides animal health and behaviour weremonitored every two days All experiments were approvedby the Ethics Committee of Zhangye People™s Hospital toguarantee that all studies involving experimental animalswere performed in full compliance with National Institutesof Health Guidelines for the Care and Use of LaboratoryAnimals The U251 — cells100 μL cells were transfected with pcDNA31IRAK3 and then transferred to micen12 Normal U251 cells were transferred to mice in the5azadC and PD325901 groups and then 5azadC andPD325901 were subcutaneously injected respectively intothe posterior flank of nude mice After and days™culture the mice were sacrificed and the tumor size wasmeasured The tumor volume was measured using a caliperfollowing the formula length — width22Statistical AnalysisAll data were collected from three independent experiments and presented as mean ± standard deviationStatistical analyses were performed using GraphPad software Differences between groups were analyzed usingStudent' ttest or chisquare test Statistical significancewas set at P ResultsGenome Methylation Profile in GliomaA total of glioma tissues and adjacent normal sampledata publicly available at GEO were analyzed to revealthe global DNA methylation patterns of glioma Firstdensity plots of β values generated from each samplewere used identifying a poor performance of methylatedsignals for raw data Figure 1A Infinium Type I and TypeII probe assays also showed somewhat different distribution of β value ranging from unmethylated sites to fully methylated sites Figure 1B Therefore these datawere regulated by normalization procedures to reduce thepotential impact later Multidimensional scaling MDSplot and dendrogram exhibited a differential clusteringbetween tumor and normal groups which distinguishedglioma from adjacent normal tissues Figure 1C and DFurther heatmap of the top differentially methylatedCpG sites indicated a visible difference in DNA methylation profiles between the tumor and normaltissueand general hypermethylation occurred insamplesglioma Figure 1EDistribution of Genomic Regions forDifferentially Methylated CpG SitesBased on the position relative to gene [1st exon ² untranslated region UTR ² UTRs body transcription start sites bp TSS1500 and TSS200] as well as CpG islands andneighborhood content shores shelves islands and sea the distribution of genomic regions for the differentiallymethylated CpG sites was exhibited More than and methylation differences occurred in bodyIGR and CpGisland sea respectively which was obviously higherthan that in other regions Figure 2A and B From thesubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related gene expression level A Based on the differential genes in the disease data the distribution of upregulated genes anddownregulated genes in the GOrelated pathway was enriched B The expression level of top MAPK signaling pathway“related genesAbbreviation MG malignant gliomaperspective of both genetics and epigenetics gene and CpGcontent regions were combined for more analyses whichshowed that CpG sites in genetic bodyisland sea andIGRisland sea were differentially methylated betweenglioma and adjacent normal samples Figure 2CIRAK3 Was Hypermethylated in GliomaThe heatmap was used to present the top hypermethylationgenes in glioma compared with normal tissues so as to figureout the most characteristic gene with methylation change intumors indicating that the IRAK3 was hypermethylated theCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressmost in glioma Figure 3A Then the methylation differentialdistribution of the IRAK3 was upregulated at each siteFigure 3B Furthermore the β value of CpG sitesIRAK3such as cg cg cg cg cg cg cg andso on on the IRAK3 was significantly higher in the tumorindicating that IRAK3group than in the normal groupwas hypermethylated in the tumor group due to CpGsFigure 3C“I The aforementioned results showed that theIRAK3 CpG sites were highly methylated in glioma tissuesthan in normal tissuestissues and the top signaling pathwayrelated genesenriched in the glioma were listed Figure 4A and B Theresults suggested that the MAPK signaling pathway washighly expressed and activated Figure 4C and D Based onthe differential genes in the disease data the distribution ofupregulated genes and downregulated genes in the GOrelatedpathway was enriched Figure 5A The expression level ofMAPK signaling pathwayrelated genes in glioma is shown inFigure 5B The results of Figures and manifested thatIRAK3related MAPK signaling pathway was highly activatedin glioma However whether any correction existed with thedevelopment of glioma remained to be verifiedMAPK Signaling Pathway Expression Leveland State in GliomaFor the identification of gliomarelated signaling pathwaysthat might be influenced by aberrant DNA methylation thetop IRAK3related signaling pathways in glioma and normalMethylation and Expression Level of theIRAK3 in Glioma Tissues and CellsThe impact of hypermethylation on the expression of IRAK3in glioma tissues and cells was elucidated The IRAK3 wasFigure Methylation level and expression level of the IRAK3 in glioma tissues and cells A The IRAK3 was highly methylated in glioma tissues compared with adjacent tissues BThe IRAK3 was less expressed in glioma tissues than in adjacent tissues C The IRAK3 in the five glioma cells SHG44 U251 HS683 SF539 and GOS3 was hypermethylatedcompared with U251 glioma cells D The IRAK3 was less expressed in the five glioma cells than in normal glioma cells The difference was significant P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Effect on glioma cells after the overexpression and demethylation of the IRAK3 A The expression level of the IRAK3 was significantly higher in the glioma U251 cells of thepcDNA31IRAK3 and 5azadC groups than in the control group B and C The protein expression level of the IRAK3 significantly increased in the pcDNA31IRAK3 and 5azadC groupscompared with the control group D The expression level of the inflammatory factor IL6 significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with thecontrol group E The activity significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantlyincreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group H and I The migration capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups compared with the control group J and K The invasiveness capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups than in the the control group Allthe mentioned differences were significant P The IRAK3 had a suppressive effect on glioma cells in vitroCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepresshighly methylated and less expressed in glioma tissues than inadjacent normal tissues Figure 6A and B Next the IRAK3 infive glioma cell lines SHG44 U251 HS683 SF539 andGOS3 also showed hypermethylation and less expressioncompared with normal gliocytes Figure 6C and D U251cells were selected for subsequent experiments because themethylation level of the IRAK3 in U251 cells was the highestFigure 6COverexpression or Demethylation of theIRAK3 Inhibited the Development of GliomaCellsA systematic test with the overexpression or demethylation ofthe IRAK3 was conducted to understand whether the correctionof abnormal IRAK3 methylation and expression affected thedevelopment of glioma After using pcDNA31IRAK3 tooverexpress or 5azadC to demethylate the IRAK3 the proteinexpression level of the IRAK3 in glioma U251 cells increasedin the tumor group compared with the normal group Figure7A“C IL6 an inflammatory factor secreted by the MAPKsignaling pathway [PMID ] so that its level in theculture medium could be used to represent the status ofMAPK signaling pathway activation decreased in thepcDNA31IRAK3 and 5azadC groups compared with thecontrol groups Figure 7D Surprisingly the viability ofglioma cells significantly decreased while the apoptosis rateincreased Figure 7E“G and the migration and invasivenesscapability decreased in the pcDNA31IRAK3 and 5azadCFigure Effect on glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The expression level of MAPKsignaling pathway“related genes expression level was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group B and C Theexpression level of MAPK signaling pathway“related proteins significantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the controlgroup D The level of inflammatory factor IL6 was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group E The activitysignificantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantly increased inthe PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group All the mentioned differences were significant P P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et algroups Figure 7H“K which elucidated the associationbetween IRAK3 and the development of glioma The aforementioned results verified that the overexpression or demethylation of the IRAK3 inhibited the development of glioma cellsin vitroMAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited the Development ofGlioma CellsThe MAPK signaling pathway inhibitors PD325901pcDNA31IRAK3 and 5azadC were used alone or incombination in vitro to confirm the influence of the IRAK3and MAPK signaling pathways on glioma In PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901groups the mRNA and protein expression levels of theMAPK signaling pathwayrelated genes such as MEK1ERK and cFos as well as the level of IL6 decreased inglioma Figure 8A“D The cell viability significantlythe apoptosis rate increased Figure 8E“Gdecreasedand the migration and invasiveness capability significantlydecreased in PD325901 pIRAK3PD325901 and 5azadCPD325901 groups Figure 9A“C The aforementioned results verified that the MAPK signaling pathwaysuppression alone or combined with the overexpression ordemethylation of IRAK3MAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited Glioma in vivoFinally whether MAPK signaling pathway suppression aloneor combined with the overexpression or demethylation of theFigure Effect on the migration and invasiveness capability of glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and5azadC A and B The migration capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group A and C Theinvasiveness capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group All the mentioned differences weresignificant P Cancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Effect on mice after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The tumor in the transplantationPD325901 p IRAK3 PD325901 and 5azadC PD325901 treatment groups after and days B The tumor volume was smaller in the PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901 groups compared with the control group C The tumor weight followed the same trend as the volume D Theexpression level of the IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups compared with thetransplantation control group E and F The protein expression level of IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups than in the transplantation control group All the mentioned differences were significant P P IRAK3 had a suppressive effect on glioma in vivo was studiedAfter transplanting glioma U251 cells treated with PD325901pcDNA31IRAK3 PD325901 or 5azadC PD325901the tumorigenesis significantly decreased compared with thatin the control group mice Figure 10A“C Finally mRNAand protein expression levels ofthe MAPK signalingsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alpathwayrelated genes in xenograft glioma were detectedthey were found to be significantly decreased in thePD325901 pcDNA31IRAK3 PD325901 or 5azadC PD325901 group Figure 10D“FDiscussionAccording to the World Health anization the overallsurvival of patients with cancer has increased significantlyover the years with the improvement in diagnosis and treatment However glioma is still a highly fatal disease1Therefore this study aimed to elucidate the detail of itsprogression mechanisms and search for new therapeutic strategies One recent study reported that the use of MGMTpromoter methylation test in hospitals had a strong influenceon the prognosis of glioma17 suggesting a significant clinicalapplication prospect of DNA epigenetic regulation Hencethe main focus of the present study was on the relationshipbetween DNA methylation and glioma IRAK3IRAK3 belongs to the IL receptorassociated kinaseIRAK family involved in inhibiting Tolllike receptorsignaling by changing the activity of other members ofthe IRAK family to decrease inflammatory response1819Increasing evidence supported thatthe expression ofIRAK3 in tumorassociated macrophages led to compromised immune surveillance of cancer cells when it profitably prevented excessive inflammation contributing tocancer development Therefore the growth of transplantable cancer cells could be inhibited by enhancing hostimmune responses in IRAK3deficient mice20 A smallnumber oftumor cellintrinsicIRAK3 could also support the progression of tumor cellsin colorectal and lung cancers depending on the dysfunctional innate immune system indirectly Interestingly theexpression of IRAKM in colorectal and lung cancers correlated with poor prognosisin patients with thesecancers2122 However little is known about the molecularmechanism of the IRAK3 in glioma This study revealedthe downregulation of IRAK3 glioma tissues and cellsthrough DNA methylation analysis which seemed to becontrary to the previous findings on other cancers Thisstudy investigated whether a special signaling pathwayexisted that connected IRAK3 and glioma progressionshowed thatstudiesAn ongoing study has validated that all members of theIRAK family mediate the activation of MAPK and nuclearfactorκB NFκB signaling pathways23 MeanwhileIRAK3 a general negative regulator was confirmed toinhibit MAPK and NFκB activation2425 Likewisethese results identified that the MAPK signaling pathwaywas highly activated in glioma and its related geneexpression level was downregulated after overexpressionof the IRAK3 In fact upstream genomic events andordifferent extracellular stimuli could activate the MAPKsignaling pathway mediating a wide range of cellularprocesses26 The activation of the MAPK signaling pathway often led to abnormal cell growth and tumorigenesisThe predominant effect relied on the signal intensity andthe context or tissue in which the signal occurred2728Zhang revealed that aberrant DNA methylation inthe promoters of MMPTIMP axis genes upregulated theMAPK signaling pathway promoting the progression oftumor cells Correction of the abnormal DNA epigenotypes attenuated the migration and invasion of tumorcells in vitro and reduced tumorigenicity in vivo29Intriguingly these results were consistent with those ofthe present studyConsidering the reverse expression pattern of IRAK3between glioma and other cancers2122 different epigeneticmodification was regarded as the major reason causing thisdistinction In many cancers the DNA methylation patternsbecame aberrant with tissue specificity serving as diagnosticmarkers and therapeutic targets30“ Hence based on thelower expression and the tumorigenic function of the IRAK3in glioma its epigen
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" leukotriene receptor antagonists ltras are broadly used for the management of allergic asthmaand have recently been indicated to inhibit carcinogenesis and cancer cell growth in colorectal cancer crcchemoprevention studies the occurrence of adenoma or crc itself is generally set as the trial endpoint althoughthe occurrence rate of crc is the most confident endpoint it is inappropriate for chemoprevention studies becausecrc incidence rate is low in the general population and needed for longterm monitoring aberrant crypt fociacf defined as lesions containing crypts that are larger in diameter and darker in methylene blue staining thannormal crypts are regarded to be a fine surrogate biomarker of crc therefore this prospective study was designedto explore the chemopreventive effect of ltra on colonic acf formation and the safety of the medicine in patientsscheduled for a poly resection as a pilot trial leading the crc chemoprevention trialmethods this study is a nonrandomized openlabel controlled trial in patients with colorectal acf and polypsscheduled for a polypectomy participants meet the inclusion criteria will be recruited and the number of acf inthe rectum will be counted at the baseline colonoscopic examination next the participants will be assigned to theltra or no treatment group participants in the ltra group will continue mg of oral montelukast for weeksand those in the no treatment group will be observed without the administration of any additional drugs at theend of the 8week ltra intervention period a polypectomy will be conducted to evaluate the changes in thenumber of acf and cell proliferation in the normal colorectal epithelium will be analyzeddiscussion this will be the first study to investigate the effect of ltras on colorectal acf formation in humanstrial registration this trial has been registered in the university hospital medical information network uminclinical trials registry as umin000029926 registered november keywords colorectal cancer chemoprevention leukotriene receptor antagonist aberrant crypt foci correspondence takuma_hyokohamacuacjpdepartment of gastroenterology and hepatology yokohama city universityschool of medicine fukuura kanazawa yokohama kanagawa japan 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 0chigurashi bmc cancer page of cancer is a one of the major health issues and a leadingcause of death globally the incidence prevalence andmortality rates of colorectal cancer crc continue torise all over the world [ ] the majority of crc casesare derived from adenomatous polyps and their resection has been shown to reduce the risk of the futuredevelopment of advanced adenomas and crc [ ]thereby preventing crcrelated deaths however patients with polyps adenomatous polyps andor hyperplastic polyps represent a highrisk group for theoccurrence of metachronous colorectal polyps andorcrc then a paradigm is shifting from surveillancefor the early detection of advance adenomas or crcsand resection to novel tactics for prevention is requiredto reduce the mortality rate of this disease a number oflargescale epidemiologic andor clinicaltrials haveassessed the prophylactic agentsincluding vitamin dcalcium fiber and nonsteroidal antiinflammatory drugsnsaids such as aspirin and selective cyclooxygenase cox2 inhibitors in preventing against crc development we previously reported that the nsaidsulindac suppressed the development of sporadic colorectal adenomas to this point nsaids particularlycox2 inhibitors have been proven to offer the greatestpossibility for reducing crc risk either alone or in combination with other drugs while it has been reportedan elevated risk of serious cardiovascular events relatedwith the administration of cox2 inhibitors [ ] inview of these adverse cardiovascular effects and the lackof efficacy of other drugs that initially looked promisingthe development of novel agents that meet both safetyand efficacy in preventing crc is essentialleukotriene receptor antagonists ltras such asmontelukast and zafirlukast are commonly used for thetreatment of allergic asthma [ ] and tsai mj reported that ltras reduced cancer risk in a dosedependent manner in asthma patients it was alsoreported that the cancer incidence rate was significantlylower in ltra users than in nonltra users vs per patientsyear this means that apart fromits role in asthma ltra has also been associated withcarcinogenesis and tumormediated immunosuppression for example the overexpression of cysteinyl leukotriene receptor cyslt1r has been observed in crcand montelukast leads the apoptosis of crc cancer cells[ ] previous in vivo studies have shown the chemopreventive effect of ltras [ ] but the chemopreventive effects of ltra have not been studied in clinicalpractice therefore we designed this study to ivestigatethe chemopreventive effect of ltras in clinical practicein crc chemoprevention trials the occurrence of adenomas or crc itself is generally set as the trial endpointthough the occurrence of crc is the most confidentendpoint it is not recommended for chemopreventionstudies because crc incidence rate is low in the generalpopulation and needed for longterm monitoring furthermore there are ethical concerns about conductinglongterm trials to determine whether a test agent is effective or notaberrant crypt foci acf defined as lesions containing crypts that are larger in diameter and darker inmethylene blue staining than normal crypts [“] areregarded to be a fine surrogate biomarker of crc our group has previously reported that acf is usefulbiomarker for crc [ ] and study endpoint for achemoprevention study [ ] the advantages of chemoprevention studies with the number of colorectalacf as the trial endpoint are that longterm observationis not needed to investigate the agent efficacy and thenumber of acf can be quantitatively estimated therefore we set the acf count as a good endpoint for thisstudy to the knowledge of us this is the first clinicalstudy investigating the use of ltras as chemopreventive agents against colorectal acf in humansmethodsstudy design and settingthis study was designed as a nonrandomized openlabel controlled trial to be conducted in patients withcolorectal acf it will be performed at the departmentof gastroenterology and hepatology yokohama cityuniversity ycu hospital japan the coordinating office will be at the ycu hospital and patient registrationwill be conducted at the ycu center for novel and exploratory clinical trials ynext and data collectionwill be done using electronic data caputureethical considerations and trial registrationthe trial protocol complies with the declaration ofhelsinki and the ethical guidelines for clinical research of the ministry of health labour and welfarejapan ethical approval of this trial was obtainedfrom the ethics committee of ycu hospital on may the study protocol and informed consent documents were approved by the ycu hospital ethics committee this trial has been approved in the clinical trialact in japan and registered in the japan registry of clinical trials jrct as jrcts031180094 and the universityhospital medical information network umin clinicaltrials registry as umin000029926 all study participants will submit a written study participation informedconsent formparticipation criteriawe will recruit the patients with colorectal acf and resectable polyps for this trial the inclusion criteria are asfollows patients with resectable polyps [adenoma 0chigurashi bmc cancer page of hyperplastic polyp and sessile serrated adenomapolypssap] patients with more than five rectal acfand submit written study participation informed consent formthe exclusion criteria are as follows patients withlesions suitable for early removal a history of ltrause within months before study participation a history of malignant disease within years before studyparticipation a history of heart renal liver failure orliver cirrhosis a history offamilial adenomatouspolyposis hereditary nonpolyposis crc or inflammatory bowel disease pregnancy or the possibility ofpregnancy prohibitions of montelukast allergiesto montelukast regular use of nsaids metforminand pioglitazone and participants considered as unsuitable for the study by the researchersinterventionall eligible participants will be assigned to the ltra orno treatment group because this is an openlabel trialpatients will be assigned to the no treatment group afterthe inclusion of patients in the ltra group participantsin the ltra group will receive mg of oral montelukast for weeks and those in the no treatment groupwill be observed without the administration of any additional drugs at the end of the 8week ltra treatmentperiod a polypectomy will be performed to evaluate thechanges in the number of acf and cell proliferation inthe normal colorectal epithelium will be analyzedoutcome measurementsthe primary endpoint will be the change in the numberof colorectal acf after weeks of treatment a magnifying colonoscope pcfq290azi hz290 olympus cotokyo japan will be used in all cases procedure preparation for the colonoscopy will begin day before theprocedure each participant will be informed to take alowresidue diet and mg oral sodium picosulfate onthe evening before the procedure on the day of the procedure each participant will be given ml polyethylene glycol peg if the stools are not clear enough anadditional ml peg will be given to ensure adequatebowel cleansing in most cases conscious sedation withmidazolam “ mg and pentazocine “ mg willbe use at the start of the colonoscopy subcutaneousscopolamine or glucagon will be administered for colonic movements reduction at the time of the first colonoscopy the endoscopists will insert into the cecumand the observe entire colorectum as the endoscope ispulled back one colonic mucosal sample will be collected the number of rectal acf will be counted usinga magnifying endoscope at the end of the 8week ltratreatment period the same endoscopists will performthe polypectomies and countthe number of acfendoscopists will record all procedures on a hard diskdrive and take photograph all acf the number of acfin each participant will first be counted by the endoscopists during the procedure to provide additional validation the number of acf will be recounted by threeblinded expert endoscopists aj ht and ak by observing the recorded hard disk drive cases that these evaluators deem colonoscopy to be inappropriate will beexcluded from the final analysisthe secondary outcomes will be as follows drugsafety adverse events aes will be graded according tothe national cancer institute common toxicity criteriafor adverse events ncictcae version all trialparticipants will be provided with a trial record for thedaily dose of the trial agent and aes participants whodevelop serious ae of grade or higher will be discontinued at that time in the study the effects ofltras on cell proliferation in the rectal mucosa onecolonic mucosal sample will be collected from the samestudy patient by performing a biopsy at the time of thebaseline colonoscopy and polypectomy a biopsy will beobtained from all participants cell proliferation will beevaluated by ki67 staining briefly we will randomly select six crypts and count the number of ki67positivecells per crypt in total cells will be counted at amagnification of × using a brightfield microscopethe results will be presented as the percentage of ki67positive cells all participants will receive laboratory testsand a physical examination at the point of the baselinecolonoscopy and polypectomydrug supplymontelukast capsules will be purchased from kyorincorporation ltd tokyo japan participants will be informed to take one tablet of the study drug every nightbefore bed medication adherence will be monitored bycounting the empty medication sheets returned by theparticipants at the time of their polypectomy the participants will also be interviewed and monitored to confirm thatthey have not used any prohibited drugsaspirin metformin andor other nsaids aes will bemonitored by the investigator and graded according tothe ncictcae version if serious aes or less than drug adherence are confirmed in a participant thisparticipant will be withdrawn from the trialsample size estimation and allocationwe previously reported the administration of mgmetformin per a day for weeks reduced the number ofacf in that trial the mean number of acf per patientdecreased significantly from ± at baseline to ± at weeks p although this study is exploratory research and the accurate chemopreventive efficacy of montelukastis unknown we assume that 0chigurashi bmc cancer page of montelukast may have an effect that is equivalent to of that observed for metformin on the reduction of acfnubmer therefore we estimate that the acf numberwill change by about ˆ’ to ˆ’ on average we determined that a sample size of “ individuals in theltra group was needed to detect a significant reduction in the number of acfs in the ltra group using apaired ttest with a twosided significance level of and power assuming some dropouts we propose to recruit participants in the ltra group to confirmthat the number of acf does not change during thestudy period we propose to recruit patients in the notreatment group after consecutively accumulating patients in the ltra group therefore we propose to recruit patientsstatistical analysisthe change in the number of acf which is the primaryendpoint will be compared before and after the 8weekstudy period between the ltra and no treatment groupsby the paired ttest drug safety will be assessed by thechisquare test and the remaining results will be compared by the ttest or mann“whitney u test between thetwo groups p will be defined as statistically significant all statistical analyses will be conducted using jmp® software sas institute inc cary nc usatrial steering committee and data monitoring committeethe trial steering committee and data monitoringthe ynext thecommittee will be located atmanagement team will perform central monitoring ofthe trial status and data collection every monthstudy flowa study flow is shown in fig discussionto the knowledge of us this is the first clinical trial proposed to investigate the efficacy of ltras on colorectalacf formation ltras are broadly used for the treatment of allergic asthma and rhinitis [ ] and ltrasare reported to decrease the risk of cancer in asthma patients in a dosedependent manner previous basicresearch has reported that cyslt1r is overexpressed incrc and that montelukast induces the apoptosis ofcrc cells [ ] cyslts have recently been focusedon as significant regulators of gut homeostasis with endogenous cyslt production mediating the proliferationand survival of gut mucosal cells recent evidence focuses on the effect of leukotrienec4 ltc4 in accelerating oxidative dna damage if notadequately repaired can contribute to increase mutationrates and genomic instability dna damage andgenomic instability are major drivers of carcinogenesis cyslts also acts as leukocyte chemoattractants inaddition cyslt1 mediates th17 cell migration the storage of which associates with the progression ofinflammationassociated cancers chronic inflammation is a risk factor for cancer initiation and progression as observed in patients with inflammatory bowelfig study flow chart 0chigurashi bmc cancer page of disease furthermore leukotriene d4 ltd4 antagonists suppress chronic inflammation in a rodent modelof acute enteritis and this may be effective in preventinginflammationassociated crc ltras are leukotriene pathway inhibitors and thus they may have potentialas chemotherapy andor chemoprevention agents to reduce the effect of leukotrienes previous in vivo studieshave elucidated the chemopreventive effect of leukotriene pathway inhibitors [ ] and showed the potentialuse of ltras for chemoprevention therefore we willconduct this trial to explore the chemopreventive effectof ltras in clinical settingthis trial may have some limitations as follow firstacf are believed to be a fine surrogate biomarker ofcrc though its biological significance in humans is stillcontroversial in crc chemoprevention studies typically set the occurrence of adenomas or the crc itselfas endpoint of the study though the occurrence of crcis the most appropriate endpoint it is inappropriate forchemoprevention studies because crc incidence rate islow in the general population and needed for longtermmonitoring our group has previously reported thatacf is useful biomarker for crc and conducted a chemoprevention study for colorectal acf [ ] therefore we designed this study using the number of acf asthe primary endpoint to investigate the chemopreventiveeffect of ltras second an intervention duration of weeks may be too short to reliably detect differences between two groups since our group reported in a previous trial that oral administration of metformin for weeks reduced the number of colorectal acf in humans an intervention period of weeks should beenough to assess the changes in the number of acf ifltras have a chemopreventive effectour group previously conducted a shortterm chemoprevention study of metformin for colorectal acfand reported the preventive effect of the agent on theformation of acf then we conducted a oneyearmetformin chemoprevention studycolorectalpolyps we propose to repeat the same steps as inour metformin study for the chemoprevention studyusing montelukastforif ltras were proved to have efficacy for crc prevention the impact would be significant therefore webelieve it will be very interesting to assess whetherltras inhibit the formation of human colorectal acfabbreviationscrc colorectal cancer nsaids nonsteroidal antiinflammatory drugs cox cyclooxygenase2 ltras leukotriene receptor antagonistscyslt1r cysteinyl leukotriene receptor acf aberrant crypt fociycu yokohama city university umin university hospital medicalinformation network ssap sessile serrated adenomapolyp ncictcae national cancer institute common toxicity criteria for adverseevents peg polyethylene glycol aes adverse events ltc4 leukotriene c4ltd4 leukotriene d4acknowledgmentsthe authors would like to thank the staff for their support in recruitingeligible patients and the patients who participated in this study and theirfamily we thank cathel kerr bsc phd and melissa crawford phd fromedanz group httpsenauthorservicesedanzgroupcom for editing a draftof this manuscriptauthors™ contributionsth ja and an conceived the study th and ja equally contributed to thismanuscript ja th ka nm ty tm af and ho will perform the baselinecolonoscopies and polypectomies ja th and ka will conduct the secondcount of acf using a hard disk drive recording to ensure validity tt nm tyaf and ho will recruit participants and followup with them at an outpatientclinic the analysis and interpretation of data will be conducted by ja thand ka all authors have read the final manuscript and approved its submission for publicationfundinga grant for this research from the kanagawa institute of industrial scienceand technology kistec was awarded to th we declare that the fundingbody has no role in the design data collection analysis interpretation andwriting of the studyavailability of data and materialsthe datasets used andor analyzed during the current study will be availablefrom the corresponding author on reasonable requestethics approval and consent to participateethical approval of this trial was obtained from the ethics committee of ycuhospital on may the study protocol and informed consentdocuments were approved by the ycu hospital ethics committee this trialhas been registered in the university hospital medical information networkumin clinical trials registry as umin000029926 all study participants willsubmit a written study participation informed consent formconsent for publicationnot applicablecompeting intereststhe authors declare that they have no conflicts of interestsreceived may accepted august referencestorre la bray f siegel rl global cancer statistics ca cancer jclin “anderson wf umar a brawley ow colorectal carcinoma in black andwhite race cancer metastasis rev “vogelstein b fearon er hamilton sr genetic alterations duringcolorectaltumor development n engl j med “ winawer sj zauber ag ho mn o™brien mj gottlieb ls sternberg ss wayejd schapiro m bond jh panish jf prevention of colorectal cancer bycolonoscopic polypectomy the national polyp study workgroup n engl jmed “citarda f tomaselli g capocaccia r barcherini s crespi m italianmulticentre study group efficacy in standard clinical practice ofcolonoscopic polypectomy in reducing colorectal cancer incidence gut“zauber ag winawer sj o™brien mj lansdorpvogelaar i van ballegooijenm hankey bf shi w bond jh schapiro m panish jf stewart et waye jdcolonoscopic polypectomy and longterm prevention of colorectalcancerdeaths n engl j med “ maisonneuve p botteri e lowenfels ab fiveyear risk of colorectalneoplasia after negative colonoscopy n engl j med “author reply das d arber n jankowski ja chemoprevention of colorectal cancerdigestion “ epub oct review matsuhashi n nakajima a fukushima y yazaki y oka t effects of sulindacon sporadic colorectal adenomatous polyps gut “ 0chigurashi bmc cancer page of drazen jm cox2 inhibitorsa lesson in unexpected problems n engl jmed “ epub feb meyskens fl jr mclaren ce pelot d fujikawabrooks s carpenter pmhawk e kelloff g lawson mj kidao j mccracken j albers cg ahnen djturgeon dk goldschmid s lance p hagedorn ch gillen dl gerner ewdifluoromethylornithine plus sulindac for the prevention of sporadiccolorectal adenoma a randomized placebocontrolled doubleblind trialcancer prev res phila “scott jp petersgolden m antileukotriene agents for the treatment of lungdisease am j respir crit care med “shen z genomic instability and cancer an introduction j mol cell biol“kim hs lee g the cysteinyl leukotriene receptor cysltr1 mediates th17cell migration j immunol bernstein cn blanchard jf kliewer e wajda a cancer risk in patients withinflammatory bowel disease a populationbased study cancer “ nishikawa m hikasa y hori k tanida n shimoyama t effect of leukotrienec4d4 antagonist on colonic damage induced by intracolonic administrationof trinitrobenzene sulfonic acid in rats j gastroenterol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations dahlen se dahlen b drazen jm asthma treatment guidelines meet the realworld n engl j med “tsai mj wu ph sheu cc hsu yl chang wa hung jy corrigendumcysteinyl leukotriene receptor antagonists decrease cancer risk in asthmapatients sci rep wang d dubois rn eicosanoids and cancer nat rev cancer “ nielsen ck the leukotriene receptor cyslt1 and 5lipoxygenase areupregulated in colon cancer adv exp med biol “ burke l butler ct murphy a moran b gallagher wm o™sullivan j kennedybn evaluation of cysteinyl leukotriene signaling as a therapeutic target forcolorectal cancer front cell dev biol savari s liu m zhang y sime w sjolander a cyslt1r antagonists inhibittumor growth in a xenograft model of colon cancer plos one e73466 bellamkonda k satapathy sr douglas d chandrashekar n selvanesan bcliu m savari s jonsson g sjolander a montelukast a cyslt1 receptorantagonist reduces colon cancer stemness and tumor burden in a mousexenograft model of human colon cancer cancer lett “ roncucci l stamp d medline a cullen jb bruce wr identification andquantification of aberrant crypt foci and microadenomas in the humancolon hum pathol “ roncucci l medline a bruce wr classification of aberrant crypt foci andmicroadenomas in human colon cancer epidemiol biomark prev “ pretlow tp barrow bj ashton ws aberrant crypts putativepreneoplastic foci in human colonic mucosa cancer res “ pretlow tp o™riordan ma pretlow tg stellato ta aberrant crypts in humancolonic mucosa putative preneoplastic lesions j cell biochem suppl “takayama t katsuki s takahashi y ohi m nojiri s sakamaki s kato jkogawa k miyake h niitsu y aberrant crtpt foci of the colon as precursorsof adenoma and cancer n engl j med “sakai e takahashi h kato s uchiyama t hosono k endo h maeda syoneda m taguri m nakajima a investigation of the prevalence andnumber of aberrant crypt foci associated with human colorectal neoplasmcancer epidemiol biomarkers prev “ epub jul ohkubo h takahashi h yamada e sakai e higurashi t uchiyama t hosonok endo h taguri m nakajima a natural history of human aberrant cryptfoci and correlation with risk factors for colorectal cancer oncol rep “takahashi h yoneda m tomimoto a endo h fujisawa t iida h mawatarih nozaki y ikeda t akiyama t yoneda m inamori m abe y saito snakajima a nakagama h life stylerelated diseases of the digestive systemcolorectal cancer as a life stylerelated disease from carcinogenesis tomedical treatment j pharmacol sci “ hosono k endo h takahashi h sugiyama m sakai e uchiyama t suzuki kiida h sakamoto y yoneda k koide t tokoro c abe y inamori mnakagama h nakajima a metformin suppresses colorectal aberrant cryptfoci in a shourtterm clinical trial cancer prev res phila “the world medical association wma declaration of helsinki “ ethicalprinciples for medical research involving human subjects the ministry of health labour and welfare ethics guidelines for clinicalresearch paruchuri s mezhybovska m juhas m sjolander a endogenous productionof leukotriene d4 mediates autocrine survival and proliferation via cyslt1receptor signaling in intestinal epithelial cells oncogene “ dvash e hartal m barak s meir o rubinstein m leukotriene c4 is themajor trigger of stressinduced oxidative dna damage nat commun 0c"
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" the empirical dietary index for hyperinsulinemia edih score is a validated foodbased dietary scorethat assesses the ability of wholefood diets to predict plasma cpeptide concentrations although the edih hasbeen extensively applied and found to be predictive of risk of developing major chronic diseases its influence oncancer survival has not been evaluated we applied the edih score in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patterns after diagnosis and determine its influence on survivaloutcomesmethods we calculated edih scores to assess the insulinemic potential of postdiagnosis dietary patterns andexamined survival outcomes in a sample of stage iiii colorectal cancer patients in the nurses™ health studyand health professionals followup study cohorts multivariableadjusted cox regression was applied to computehazard ratios hr and confidence intervals ci for colorectal cancerspecific mortality and allcause mortalitywe also examined the influence of change in diet from pre to postdiagnosis period on mortalityresults during a median followup of years there were deaths which included colorectal cancerspecific deaths in the multivariableadjusted analyses colorectal cancer patients in the highest compared tolowest edih quintile had a greater risk of dying from colorectal cancer hr ci and a greater risk of allcause death hr 95ci compared to patients who consumed low insulinemicdiets from pre to postdiagnosis period patients who persistently consumed hyperinsulinemic diets were at higherrisk of colorectal cancer death hr151 95ci and allcause death hr 95ci our findings suggest that a hyperinsulinemic dietary pattern after diagnosis of colorectal cancer isassociated with poorer survival interventions with dietary patterns to reduce insulinemic activity and impactsurvivorship are warrantedkeywords colorectal cancer survival insulinemic dietary patterns insulin cpeptide correspondence fredtabungosumcedu1division of medical oncology department of internal medicine the ohiostate university college of medicine west 12th avenue 302b wisemanhallccc columbus oh usa2the ohio state university comprehensive cancer center arthur g jamescancer hospital and richard j solove research institute columbus oh 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 0ctabung bmc cancer page of canceristhefourth most colorectalcommonlydiagnosed cancer in the united states while there ishigh potential for dietary patterns as a modifiable riskfactor for colorectal cancer development [ ] verylimited evidence exists among colorectal cancer survivors for example in a recent review we identified s published up to that reported on theassociation between dietary patterns and colorectalcancer development but only about five s onthe association between dietary patterns and outcomesamong colorectal cancer survivors [“] the evidenceshowed that the western dietary pattern often characterized by high intakes ofred andprocessed meats desserts and potatoesis associatedwith higher risk of allcause mortality but generally notwith colorectal cancerspecific mortality in patients withcolorectal cancer the prudent dietary pattern oftencharacterized by high intakes of fruits vegetables wholegrains and poultry showed similar results with inverseassociations for allcause mortality but no consistent association with colorectal cancerspecific mortality [“]higher adherence to other dietary patterns including themediterranean diet score dietary approaches to stophypertension meal plan american cancer society cancerprevention guidelines score healthy eating index scorewere generally associated with lower risk of allcausemortality but the associations were inconsistent acrossstudies [ ]refined grainschronic diseasesfurther research is therefore needed to clarify ifdietary patterns are importantfor colorectal cancerprognosis and if dietary changes can maximally impactoverall and cancerspecific survival biomarkerbaseddietary patterns may be helpfulin this regard forexample hyperinsulinemia and insulin resistance areconsidered important underlying mechanisms linkingpoor dietary patterns and lifestyle behaviors to the development of multipleincludingcolorectal cancer [“] studies have shown positiveassociations between circulating cpeptide concentrations a marker of beta cell secretory activity and colorectal cancer risk and prognosis [“] therefore adietary pattern associated with hyperinsulinemia may bemore predictive of outcomes following colorectal cancerdiagnosis than a dietary pattern not associated with thispathway we previously derived the empirical dietaryindex for hyperinsulinemia edih score to assess thepotential of dietary patterns to influence insulinemia which has been extensively applied in large cohortstudies and found to be predictive of nonfasting cpeptide concentrations [ ] longterm weight gain risk of developing colorectal cancer other digestive system cancers and other cancers however the influence of dietary insulinemic potentialon cancer survival outcomes has not yet been evaluatedthe objective of the current study is to apply the edihscore in a large cohort of colorectal cancer patients toassess the insulinemic potential of their dietary patternsafter diagnosis and determine its influence on survivaloutcomesmethodsstudy populationwe used data from the nurses™ health study nhs andthe health professionals followup study hpfs twoongoing cohorts in the united states hpfs was initiatedin and enrolled male health professionalsbetween the ages of and years nhs initiatedin enrolled registered female nurses aged to years fig data on medical lifestyleand other healthrelated factors was collected at baselineand have been updated every years thereafter ethicalapproval for our study was provided by the harvardth chan school of public health and those of participating registries as required and the institutional reviewboards of the brigham and women™s hospital studyparticipants provided consent by completing and submitting study questionnaires participants were free toterminate participation in the study at any timeassessment of diet and the empirical dietary index forhyperinsulinemia edih scorein both cohorts diet was assessed using a validated selfadministered food frequency questionnaire ffq thatassessed how often on average participants consumed astandard portion size of various foods in the past yearin the nhs diet was assessed in andevery years thereafter whereas in the hpfs diet wasassessed in and every years thereafter theedih score developed to empirically measure the insulinemic potential of whole diets using food groups hasbeen described in detail briefly thirtynine foodgroups were entered into stepwise linear regressionmodels to identify a dietary pattern most predictive ofplasma cpeptide levels the edih score represents aweighted sum of food groups with higher scores indicating hyperinsulinemic diets hyperinsulinemia andlower scores indicating low insulinemic diets the foodgroups contributing to lower edih scores are winecoffee fullfat dairy products whole fruit and green leafyvegetables whereas the food groups contributing to highedih scores arelowfat dairy products french frieslowenergy beverages cream soups processed meat redmeat margarine poultry nondark fish high tomatoesenergy beverage and eggs in the current study we calculated edih scores foreach participant based on the selfadministered ffqspostdiagnosis edih score was calculated based on the 0ctabung bmc cancer page of fig flow chart describing the flow of participants from the full cohorts to the final analytic sample in the nurses™ health study nhs andhealth professionals followup study hpfsfirst ffq returned at least months but not more than years after colorectal cancer diagnosis thus avoidingdiet assessment during active cancertherapy themedian time from diagnosisto postdiagnosis dietassessment was years prediagnosis edih score wascalculated based on the cumulative average of edihscores up to the last diet assessment before colorectalcancer diagnosis the median time from prediagnosisdiet assessment to diagnosis was yearspatients with colorectal cancer and mortality assessmentwhen a colorectal cancer diagnosis was reported duringthe previous years on the followup biennial questionnaires we requested permission to obtain hospital records and pathology reports blinded study physiciansthen reviewed these records and recorded data on tumorcharacteristics for nonrespondents the national deathindex was used to identify deaths and ascertain anydiagnosis of colorectal cancer that contributed to deathafter years of followup for disease diagnoses “ in nhs and to in hpfs we identified patients with pathologically confirmed colorectalcancer we excluded participants who died before in nhs or in hpfs had reported any cancerexcept nonmelanoma skin cancer before colorectalcancer diagnosis who died at diagnosis who did nothave prediagnosis diet or postdiagnosis diet patientswho did not complete a diet assessment between months and years after diagnosis or had diet assessedoutside of this period who had diabetes at colorectalcancer diagnosis and patients with stage iv or unknownstage at diagnosis therefore the current analysis included patients with stage i ii or iii colorectal cancerincluding participants from hpfs and from nhs fig deaths were ascertained throughreporting by family for persistent nonresponders wequeried the national death index with their names up 0ctabung bmc cancer page of to june for nhs and january for hpfs cause of death was assigned by blinded physicianscovariate assessmentboth cohorts assessed covariate data eg medical historylifestyle and health factorsthrough selfadministeredquestionnaires every years these factors included physical activity smoking habits alcohol intake multivitaminuse endoscopy status regular use of aspirin and othernonsteroidal antiinflammatory drugs nsaids familyhistory of colorectal cancer weight height menopausalstatus and postmenopausal hormone use only forwomenin both cohorts as previously described dietassessment was conducted every years [ ]statistical analysiswe categorized the edih score into quintiles withcohortspecific cutoffs then pooled the data for analysispersontime of followup was calculated from the dateof postdiagnosis diet assessment to death or to lastfollowup date january in hpfs or june innhs whichever was first we used the kaplanmeiermethod to generate survival curves by quintiles of edihscore and tested group differences highest vs lowestquintile using the logrank test for this test the edihscore was adjusted for total energy intake and bmi usingthe residual methodcox proportional hazards regression was used to calculate hazard ratios hrs of colorectal cancerspecificdeath or allcause death in edih quintiles quintile cutpoints were created separately by sex and applied in thepooled sample given that participants must survivefrom diagnosis until postdiagnosis diet assessment weused time since diagnosis as the underlying time scale toaccount for left truncation due to staggered entry thecox models were tested for the assumption of proportionality using timecovariate interaction terms andstratified by age sex and stage we fitted two models tothe data as follows model minimally adjusted modelincluded bmi demographic factors sex age at diagnosis and tumor characteristics stage subsite within thecolon grade of tumor differentiation model fullyadjusted model included all the covariates in model and postdiagnosisfactors packyears ofsmoking physical activity regular aspirin use pre topostdiagnosis weight change total alcohol intake andprediagnosis dietary pattern edih score testforlinear trend of risk across edih quintiles was performedusing the median postdiagnosis edih score in eachedih quintile as a continuous variable in the cox regression models and interpreting the pvalue of thisvariable as the pvalue for linear trendlifestyleto determine how changes in the insulinemic potential of diet before and after diagnosis influence survivalwe dichotomized pre and postdiagnosis edih scores atthe median and used to create a change variable withlow indicating a score below the median and high ascore above the median lowlow consistently lowdietary insulinemic potential before and after diagnosisie both scores below the median lowhigh patientsconsuming low insulinemic diets before diagnosis andmore hyperinsulinemic diets after diagnosis highlowpatients consuming hyperinsulinemic diets before diagnosis and then changed towards low insulinemic dietsafter diagnosis and highhigh patients who consistentlyconsumed hyperinsulinemic dietary patterns before andafter diagnosis we then applied these dietary patternchanges in multivariableadjusted cox models to examine risk of death from colorectal cancer and from othercauseschange preand postdiagnosisfrom postdiagnosis weightwe conducted exploratory subgroup analysesincategories of the following potential effect modifiers sexweightand prediagnosis edih score we categorized prediagnosisedih at the median median and ‰¥ median weightchange was calculated by subtracting prediagnosisweightthe continuousweight change variable was categorized as follows thosewho gained more than kg had a stable weight ˆ’ kg to kg or lost more than kg we also conducted subgroup analyses by time since diagnosis years ‰¥ years and age at diagnosis years ‰¥years tests of interaction between postdiagnosis edihscore and the potential effect modifiers were assessed byentering in the modelthe cross product of postdiagnosis edih score and the stratification variable andevaluated by the wald test all analyses were performedusing sas for unix all pvalues were two sidedresultscharacteristics of patients women with colorectal cancer after diagnosis are shown in table meanage at diagnosis was years and mean postdiagnosisbmi was kgm2 with of patients classified asoverweight or obese regarding disease stage hadstage i or ii and had stage iii disease during amedian followup of years there were allcausedeaths which included colorectal cancerspecificdeaths median overall survival by cancer stage was years for those with stage i disease years for thosewith stage ii and years for those with stage iiidisease fortyone percent of patients maintained astable weight between ˆ’ kg and kg between theprediagnosis and postdiagnosis period while lostmore than kg body weight and gained kgbody weight in the same period colorectal cancer patients with the most hyperinsulinemic dietary patterns 0ctabung bmc cancer page of table postdiagnosis characteristics of colorectal cancer patients by quintiles of postdiagnosis empirical dietary index forhyperinsulinemia edih score n characteristictotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile quintile ˆ’ to ˆ’ to n n quintile ˆ’ to ˆ’ n quintile ˆ’ to n quintile to n female age at diagnosisdage at diagnosis by sexdfemalemalecurrent smoker packyears of smokingbody mass index kgm2overweight bmi ‰¥ obese bmi ‰¥ physical activity methweekcphysical activity methweekcd by sex femalemale nonalcohol drinkers regular aspirin use location of cancer in the colon proximal colondistal colonrectumunspecifiedstage at diagnosis stage istage iistage iiimedian survival time yearsmedian survival time by staged years stage istage iistage iiiweight change categoriesd stable weight ˆ’ to kggained more than kglost more than kg avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intakecmetabolic equivalents from recreational and leisuretime activitiesdvalue is not age adjustedafter diagnosis quintile tended to have higher bodyweight and lower physical activity for example theaverage bmi among those classified in quintile was kgm2 and the average physical activity was methourweek compared with kgm2 and methourweek among those in quintile alsopatients consuming the most hyperinsulinemic dietarypatterns were less likely to have stage i disease and theyexperienced shorter survival times table patients consuming low insulinemic dietary patternshad higher intakes of wholegrains nuts vegetables wholefruits and coffee and lower intakes of refined grainscream soup eggs french fries butter margarine sugarsweetened beverages red meat and processed meat in 0ctabung bmc cancer page of table median 5th 95th percentile food and nutrient intake profiles of colorectal cancer patients by quintiles of postdiagnosisempirical dietary index for hyperinsulinemia edih scoretotal population n quintiles of the empirical dietary index for hyperinsulinemia edih scoreabquintile n quintile n quintile n quintile n quintile n foods servingsweekprocessed meatred meat highenergy sugary beverages lowenergy sugary beverages margarinebutterfrench friesnondark fisheggslowfat dairycream souprefined grainstomatopoultrydark fishfullfat dairycoffeeteawhole fruitfruit juicepotatoesgreenleafy vegetablesdarkyellow vegetablesother vegetablesnuts total alcohol intake drinksweek whole grainsnutrient profile total carbohydrates gdtotal protein gd branchedchain amino acids gd total fat gdtotal fiber gd avalues are means sd for continuous variables and percentages for categorical variables and are standardized to the age distribution of the study populationbedih scores were adjusted for total energy intaketerms of the nutrient profile resulting from this postdiagnosis dietary pattern patients consuming a lowinsulinemic dietary pattern had higher intakes of totalcarbohydrates and total fiber and lower intakes of total fattotal protein and branchedchain amino acids table kaplanmeier curves by quintiles of edih score areshown in fig with patients consuming a lowinsulinemic diet quintile experiencing better survivalfor colorectal cancerspecific and overall mortalitycompared to those consuming hyperinsulinemic dietsquintile in the multivariableadjusted analyses wefound that a hyperinsulinemic postdiagnosis dietarypattern was associated with higher risk of colorectalcancerspecific mortality and allcause mortality table 0ctabung bmc cancer page of fig kaplan“meier curves of a colorectal cancerspecific and b overall survival among patients with colorectal cancer by quintile of postdiagnosis empirical dietary index for hyperinsulinemia edih score logrank pvalues were calculated to test group differences quintile vs and adjusted for postdiagnosis total energy intake and postdiagnosis body mass indexcomparing colorectal cancer patients classified in the highestedih quintile to those in the lowest quintile there was a higher risk of colorectal cancerspecific death hr 95ci ptrend and a higher risk of allcause death hr 95ci ptrend afteraccounting for prediagnosis dietary insulinemic potentialamong other confounding variables table in relation to changes in the insulinemic potential ofthe diet before and after diagnosis patients whoconsumed a more hyperinsulinemic dietary patternconsistently before and after diagnosis were at higherrisk of dying from colorectal cancer hr ci and from other causes hr ci compared to patients who consistentlytable hazard ratios ci for colorectal cancerspecific and allcause mortality among patients with colorectal cancer byquintile of postdiagnosis edih scorestatistical modelcolorectal cancerspecific mortalityquintiles of the empirical dietary index for hyperinsulinemia edih scorequintile quintile quintile quintile ptrendquintile deathspatients aliveminimallyadjusted model reference fully adjusted model reference allcause mortalitydeathspatients aliveminimallyadjusted model reference fully adjusted model reference the minimallyadjusted models was adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stagegrade of tumor differentiation and location of primary tumor within the colon the fullyadjusted model was additionally adjusted for postdiagnosis physicalactivity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake andprediagnosis edih score 0ctabung bmc cancer page of fig hazard ratios for the association of change in dietary insulinemic potential between prediagnosis diet and postdiagnosis diet and risk ofdying form colorectal cancer crcsurvival and from all causes combined overall survival edih scores were dichotomized at the median lowlow the reference category represents participants who persistently consumed low insulinemic diets below the median edih from pre to postdiagnosis period lowhigh are those who changed from low insulinemic diets towards more hyperinsulinemic diets highlow represents thosewho changed from consuming hyperinsulinemic diets prior to diagnosis towards consuming low insulinemic diets after diagnosis whereas highhigh represents those who persistently consumed hyperinsulinemic diets prior to diagnosis and after diagnosis the number of deaths patientsalive in the four categories were as follows crcsurvival lowlow lowhigh highlow highhigh overallsurvival lowlow lowhigh highlow highhigh models were adjusted for age at diagnosis postdiagnosisbody mass index total energy intake sex race year of diagnosis cancer stage grade of tumor differentiation location of primary tumor withinthe colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirin use weight change pre to postdiagnosis postdiagnosis total alcohol intake and prediagnosis edih scoreconsumed a low insulinemic dietary pattern before andafter diagnosis fig in subgroups of potential effect modifiers risk of colorectal cancerspecific mortality was significantly elevatedamong women and among those who lost body weightthose who were consuming a hyperinsulinemic dietarypattern before diagnosis and those younger than years for these subgroup analysesinteractions werestatistically significant only for sex in allcause mortalitytable discussionin the current study we showed that habitual consumption of hyperinsulinemic dietary patterns after colorectalcancer diagnosis or consumption of a hyperinsulinemicdietary pattern consistently before and after diagnosiswas associated with higher risk of dying from colorectalcancer and from all causes combinedthe insulinemic potential of diet was first estimatedby the insulin index which is based on a conceptsimilar to the more widely used glycemic index thatcharacterizes carbohydratecontaining foods accordingto their ability to raise blood glucose concentrationspostprandially compared with a reference food glucoseor white bread though carbohydrate content isone important factor influencing insulin response foodscan also stimulate insulin secretion in a carbohydrateindependent manner the insulin index directly quantifies the postprandial insulinemic potential of a food andtakes into account foods with a low or no carbohydratecontent it is important to understand that theinsulin index which was used in most previous studiesof dietary insulinemic potential and colorectal cancersurvival is conceptually and technically different fromthe edih and essentially uncorrelated spearman r ˆ’ the principle ofthe insulin index is how aparticularfood item stimulated insulin secretionindependent of underlying insulin resistance whereasthe edih is primarily driven by insulin resistance forcolorectal cancer the only other paper using the edihwas on cancer incidence both the insulin index and glycemic index assess thepostprandial shortterm effects of the diet unlike theedih score which predicts integrated insulin exposureie both fasting and nonfasting based on habituallongterm dietary intake postdiagnosis insulinindex and insulin load have been linked to higher risk ofdying from colorectal cancer [ ] higher dietary insulin load and insulin index after diagnosis of colorectalcancer were associated with increased risk of colorectalcancerspecific and overall mortality the association of postdiagnosis glycemic indices with colorectal 0ctabung bmc cancer page of table subgroup analyses of the association between dietary insulinemic potential and colorectal cancerspecific and allcausemortalitysubgroupptrendpinteractionquartile quartile quartile deathspatientsaliveedih quintilesquartile colorectal cancerspecific mortalitysexmenwomenweight change post minus prediagnosis weightstable weight ˆ’ to kgweight gain kgweight loss kgprediagnosis edih score median‰¥ medianage group at diagnosis years‰¥ yearstime since diagnosis years‰¥ yearsallcause mortalitysexmenwomenweight change post minus prediagnosis weightstable weight ˆ’ to kgweight gain kgweight loss kgprediagnosis edih score median‰¥ medianage group at diagnosis years‰¥ yearstime since diagnosis years‰¥ years ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref ref models were adjusted for age at diagnosis postdiagnosis body mass index total energy intake sex race year of diagnosis cancer stage grade of tumordifferentiation and location of primary tumor within the colon postdiagnosis physical activity postdiagnosis pack years of smoking postdiagnosis regular aspirinuse weight change pre to postdiagnosis and postdiagnosis total alcohol intake and prediagnosis edih scorecancer prognosis has been inconsistent whereas onestudy found higher risk of colorectal cancer recurrenceand death associated with higher glycemic load but nothigher glycemic index another found no associationbetween glycemic load or glycemic index and colorectalcancer survival glycemic scores are primarilyreflective ofthe postprandial glucose responses ofcarbohydratecontaining foods whereas the edih scoredirectly reflects insulin increases induced by componentsof the dietary pattern that may or may not be contributing to calories eg coffee current study findingstherefore suggest that the direct effect of the diet on 0ctabung bmc cancer page of insulin may be more important than the effect of diet onglucose for colorectal cancer prognosis though theglycemic index is a measure of the shortterm postprandial effect of the diet on glucose concentrations it ispossible that such a habitual dietary pattern could overtime lead to sustained hyperinsulinemia and insulin resistance which could then mediate colorectal cancerprognosis however a previous study in these cohortsdid not observe an association between an overall lowcarbohydrate diet score and colorectal cancer or overallmortality although those who consumed a plantrichlowcarbohydrate diet which emphasized plant sourcesof fat and protein with moderate consumption of animalproducts had lower risk of colorectal cancerspecificmortality insulin is a growth factor and major regulator of cellmetabolism and its effects in target cells are mediatedby the insulin receptor a transmembrane protein withenzymatic activity evidence suggest that insulinstimulates growth mainly via its own receptor and notthe igf1 receptor and that in many cancer cells theinsulin receptor is overexpressed and the a isoformwhich has a predominant mitogenic effectis morerepresented than the b isoform the metabolicpathway stimulated by the activated insulin receptor toregulate glucose protein and lipid metabolism involvesthe pi3kakt pathwaycharacteristicsprovide a selective growth advantage to cancer cellswhen exposed to insulin therefore all conditions ofhyperinsulinemia both endogenousdiabetes metabolic syndrome obesity and exogenouseg hyperinsulinemic diets which also influence someof the endogenous conditions [ ] willincreasecancer risk and mortality theseegtypefor most ofalthough interactionsthe subgroupanalyses were not statistically significant some of thefindings merit some discussion the associations werestronger among women than among men which may berelated to severalfactors the larger sample size andstatistical power in our evaluation of women potentialconfounding with age as women were younger on average than men and a true biological interaction basedupon endocrine and associated metabolic factors wealso observed that there were worse outcomes amongpatients who lost weight than among those who maintained a stable weightto postdiagnosisperiod which may be consistent with complications ofprogressing disease leading to poor diet intakefrom premajor strengths of our study include the use of afoodbased edih score that is correlated with circulating cpeptide concentrations [ ] we had access tocomprehensive pre and postdiagnosis data on diet andimportant covariates which reduces the potential for residual confounding and recall bias our findings alsoaccounted for potential bias from staggered entry due todifferences between participants in the time betweendiagnosis and postdiagnosis diet assessment limitations to be considered in interpreting our findings include potential measurement error in the selfreporteddietary and lifestyle data though prior studies in thehpfs and nhs that evaluated the relative validity offfq data have shown reasonably good correlations between ffq and diet records [ ] though we adjusted for several potential confounding variables ahyperinsulinemic dietary pattern may be associated withother factors not included in the current study therefore we cannot completely rule out confounding byunmeasured variables given that we did not have information on cancer treatment which could influence dietary choices of cancer patients or modify the diet andsurvival association we adjusted all analyses by cancerstage at diagnosis which is the principal determinant ofcolorectal cancer treatmentin this large prospective study a higher edih scorereflecting higher insulinemic potential of the diet wasassociated with higher risk of death from colorectalcancer and from all causes taken together our resultssuggest that this association may be mediated partlythrough mechanisms involving hyperinsulinemia interventions with dietary patterns to reduce insulinemia mayenhance survivorship among colorectal cancer patientsabbreviationsbmi body mass index ci confidence interval edih empirical dietary indexfor hyperinsulinemia score ffq food frequency questionnairehpfs health professionals followup study hr hazard ratio methourweek metabolic equivalent hours per week nhs nurses™ health study nsaids nonsteroidal antiinflammatory drugs pi3kakt phosphatidylinositol kinaseprotein kinase b sas® statistical analysis software®acknowledgementswe would like to thank the participants and staff of the nurses™ health studyand health professionals followup study for their valuable contributions asw
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99mtc‘labeled nanocolloid drugs development methodsVladimir Sadkin1 Viktor Sкuridin1 Evgeny Nesterov1 Elena Stasyuk1 Alexander Rogov1 Natalya Varlamova1 Roman Zelchan2The work considers the problem of obtaining nanocolloid radiopharmaceuticals RPs and studying their functional suitability for diagnosing sentinel lymph nodes SLN in cancer patients Two principal approaches to the formation of technetium99mlabeled ps based on inanic and anic matrices were considered when carrying out research to develop methods for the production of nanocolloid RPs The composition of the reagents and the conditions for obtaining nanocolloid radiopharmaceuticals were determined The functional suitability of new RPs for scintigraphic diagnostics of sentinel lymph nodes has been studiedThe identification of sentinel lymph nodes”the first nodes that stand in the way of metastasizing of malignant neoplasms attracts increasing interest in modern oncological practice1“ It is believed that if the SLN is not affected by the metastatic process then all other regional lymph nodes are intact so the results of biopsy of these nodes are an objective diagnostic criterion for the spread of malignant process Fig a0 The optimal method of detecting SLN is the use of colloid nanomaterials labeled with technetium99m for scintigraphic or radiometric determination of node localization6“ Not so much the chemical nature of such ps but their size is the determining factor in the choice of the indicator in this case Thus according to Schauer14 a colloid with a p size of less than a0nm can accumulate not only in the SLN but also at nodes of and orders of magnitude Ps with the sizes of more than a0nm slowly migrate from the injection site The colloid with the p size from to a0nm was recognized as the optimal one for detecting SLNThe simplest method of obtaining colloids with given sizes and properties is immobilization of 99mTc on the surface of nanosized materialsTechnetium99m is by far the most popular radionuclide for conducting diagnostic studies practically in all fields of medicine15“ This is primarily due to its nuclearphysical characteristics a relatively short halflife a0h and Îradiation energy of a0meV providing a low exposure dose and at the same time sufficient penetrating power for radiometric measurementsToday the Tc99m Tilmanocept radiopharmaceutical is widely used which has proven itself well and gives good results But its production is quite timeconsuming and requires expensive components We offer a less laborious method from the simple components1920Materials and methodsMaterials All the reagents were purchased from SigmaAldrich ACS grade and used without further purification Technetium99m was obtained from chromatographic 99Mo99mTc generator œ99mTcGTTOM produced by Tomsk Polytechnic University TPU”Tomsk RussiaThree types of nanops were selected to obtain nanocolloids labeled with 99mTc The first type of colloids was created on the basis of metal chelates with chemically modified complexons of diethylenetriaminepentaacetic acid DTPA It should be noted that the DTPA molecule itself like its complexes with metals is hydrophilic and does not tend to form colloidal ps The introduction of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified DTPA complexes21 The original substance of the modified DTPA DTPAmod was synthesized in Tomsk Polytechnic University Preparation of colloid solution DTPAmod was produced using the following method A sample of modified DTPA with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of NaHCO3 solution by heating to a0°C After that the volume was adjusted with the same solvent up to the mark In order to reduce the p size the container with suspension was heated in water to a0°C and treated with ultrasound for a0min 1Tomsk Polytechnic University Lenina Avenue Tomsk Russia 2Tomsk National Research Medical Center Russian Academy of Sciences Kooperativny street Tomsk Russia email sadkintpuruScientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorFigure a0 The general scheme for the synthesis of 99mTcDTPAmodwhich reduced the average p radius up to a0nm The general scheme for the synthesis of 99mTcDTPAmod is shown in Fig a0The second type of colloids is iron nanops coated with a carbon shell of FeC Fig a03a These ps were obtained from the Institute of Metal Physics UrB RAS Ekaterinburg Russia In order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates ADT onto the surface of these ps has been developed An effective method for the synthesis of ADT followed by their application onto the carbon surface of ps was developed at the Tomsk Polytechnic University22 The general scheme for the synthesis of FeC ps and their subsequent interaction with 99mTc is shown in Fig a03bIn the third type of colloids technetium99m was adsorbed on aluminum oxide powder A powder of lowtemperature cubic modification of gammaoxide Al2O3 prepared from aluminum hydroxide powder AlOH3 by its calcination in a muffle furnace was used The substance was synthesized in Tomsk Polytechnic UniversityA reducing agent”tin II chloride dihydrate was used in order to obtain complexes of 99mTc with colloidsGelatin powdered Ph Eur USPNF pure pharma grade CAS Number was purchased from AppliChem GmbH Darmstadt GermanyMethods Method for preparation of 99mTc labeled nanocells The introduction of the radioactive label 99mTc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent SnCl2ˆ™2H2O “ a0mgml in different ratios and then adding a a0ml of eluate of 99mTc “ a0MBqml to the mixtures The mixtures were incubated for a0min at a temperature of “ a0°C The preparation is ready for use after cooling at room temperature The reducing agent SnCl2ˆ™2H2O was used as a hydrochloric acid solution The amount of a0g of tin chloride II is added to the vial and a0ml of a0M hydrochloric acid HCl Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Carbon encapsulated iron nanop B the general scheme for the synthesis of FeC psis then added for its preparation After dissolution the volume is adjusted with distilled water to a0ml Dissolution was carried out in an inert gas argon mediumDetermination of the size of 99mTc labeled colloidal ps The determination of the size of the labeled nanocolloids was carried out by spectroscopy on a Nanophox p size analyzer œSympatec GmbH Germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm Three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity Calculations of the yield of products with different p sizes were determined according to the formulas given belowC220 Avc ˆ’ A1Avc C100 A1 ˆ’ A2A1 C50 A2 ˆ’ A3A2where Avc is the activity of the initial suspension prior to filtration A1 is the activity measured after filtration through a a0nm filter A2 is the activity after filtration through a0nm filter A3 is the activity measured after filtration through a0nm filterIn parallel determination of the radiochemical purity RCP of preparations by thin layer chromatography method was carried outThin‘layer chromatography TLC procedure To determine radiochemical purity of 99mTc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip Sorbfil Russia — a0 cm To determine Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cAmount of SnCl2ˆ™2H2O mgA[Sn99mTc]A ATcVIIA Table Change in relative activities of the complex [Sn99mTc] and 99mTc VIIpertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min In this system pertechnetate migrated with the front of the mobile phase Rf To determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min In this system the 99mTc“nanocolloid migrated with the front of the mobile phase Rf Stability The stability of 99mTc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mTc“nanocolloid following by incubation at a0°C for a0h At different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using TLC24Determination of the functional suitability of preparations for scintigraphic detection of SLN A study to assess the functional suitability of new nanocolloid RPs was performed in series of experiments involving white Wistar male rats weighing “ a0g Injection of RP in a dose of “ a0MBq was performed between the first and second fingers of the rat™s hind paw The animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study Since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix Static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ECAM Signature gamma camera Siemens Germany The results of scintigraphic studies determined the percentage of accumulation of RP in the lymph node and the injection site The maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œEuropean Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes Strasbourg The experimental protocols were approved by Cancer Research Institute Biomedical Ethics Committee Protocol number All invasive manipulations with animals were performed using inhalation or drug anesthesiaStatistical analysis All mean values are expressed as IDg ± SD Data were analyzed statistically using methods of general statistics with a commercially available software package œStatistics for Windows StatSoft Inc Version Results and discussionTo carry out the labeling of colloids 99mTc extracted from a standard generator in the form of pertechnetate ions contained in a NaCl solution was used It has a higher degree of oxidation VII in this chemical form and is not prone to complex formation A reducing agent”tin II chloride dihydrate widely used for the preparation of various compounds labeled with 99mTc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 As a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess SnCl2·2H2O or the additional formation of a complex of reduced 99mTc with tin26 All this required preliminary experimental studies to establish the necessary and sufficient amount of SnCl2·2H2O in the reaction mixtureDuring the experiments it was found that the optimal concentration of Sn II in the composition of the reaction mixture when it interacts with 99mTc should be in the range of “ a0mgml Table a0It was found that when the eluate with the preliminarily reduced 99mTc VII was added to the nanops the Sn II concentration introduced in the RP was CSn a0mgml almost the entire amount of 99mTc has time to enter the composition of the largesize complex with tin even before its mixing with nanops This means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the Sn II solution into the reaction mixture In this connection the reduction of 99mTc with tin II was carried out in the presence of the selected substance In this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex The technique of applying of the 99mTc label to the surface of nanosized ps is given in the previous sectionAs a result of the studies reagent compositions and conditions for obtaining three nanocolloid RPs were determined Table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmProceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mTc VII in the obtained preparations is “ Preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm Scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in Fig a0Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cComposition of the preparation per a0mlDTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Al2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Colloid yield “ a0nm RCP ± ± ± Table Composition of reagents for production of technocium99 a0m nanocolloidsFigure a0 Distribution of the preparation in the rat when the preparation is administered [Al2O3 99mTc Sn II] A immediately after the administration of the drug B a0min after the administration C a0min after the administrationComposition of preparations per a0mlAl2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n DTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n Yield of colloid “ a0nm RCP ± ± ± Table Indicators of RCP and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsScintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mTc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid Along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed Gelatin G was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system Matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mTc The results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm Table a0In addition the size of these ps was determined on a Nanophox p analyzer The obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in Fig a0 A B C The average p size diameter is and a0nm respectivelyStability test showed that complex 99mTc“nanocolloid was stable in normal serum at least for a0h Radiochemical purity of the tracer at the end of the experiment was ± A study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes Fig a0 Table a0 displays the Al2O3 99mTc DTPAmod 99mTc FeC 99mTc biodistribution data at different time points postinjectionThe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionAs a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid RPs were determined An experimental dependence of the change in the content of 99mTc VII impurities on the concentration of tin II was established and its minimum amount a0mgml was determined to reach a RHP greater than In this case the yield of the target colloid with p sizes of ± a0nm is “ Preliminary tests of the developed preparations on experimental animals showed that accumulation of RP in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range Increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml In addition there was an increase in the yield of the colloid Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Change in the density of the distribution of the number of ps from their size in radiopharmaceuticals A œ99mTcAl2O3 B œ99mTcFeC C œ99mTcDTPAmodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ Repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cStomachTime h99mTcAl2O399mTcDTPAmod99mTcFeC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figure a0 Distribution of the preparation in the rat with injection of suspension [Al2O3 99mTc Sn II Gelatin] A immediately after the administration of the preparation B a0min after the administration C a0min after the administration D a0min after the administration The numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± BloodmlHeart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table Biodistribution data up to a0h after injection of “ a0MBq of 99mTc in healthy male rats Data represent the average value n accumulation in the SLN Thus the level of accumulation of RP œ99mTcDTPAmod and RP œ99mTcFeCADT in the SLN is and respectively At the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityReceived March Accepted July References Jakobsen J K Sentinel node biopsy in urooncology A history of the development of a promising concept Urol Oncol “ Weixler B et al Sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival A prospective singlecenter trial World J Surg 101007s0026 Beasley G M et al Sentinel Lymph node biopsy for recurrent elanoma A multicenter study Ann Surg Oncol Moser J et al Sentinel node biopsy in melanoma A singlecentre experience with consecutive patients Br J Dermatol 101245s1043 “ Buda A et al Optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer Comparison of realtime fluorescence with indocyanine green and methylene blue Int J Gynecol Cancer “ Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c“ Sahbai S et al Pericervical injection of 99mTcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in SPECTCT Clin Nucl Med “ Hoogendam J P et al 99mTcnanocolloid SPECTMRI fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer J Nucl Med “ Stoffels I Leyh J P¶ppel T Schadendorf D Klode J Evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies Prospective randomized clinical trial to compare ICG99mTcnanocolloid hybrid tracer versus 99mTcnanocolloid Eur J Nucl Med Mol Imaging “ Beisani M et al Initial experience in sentinel lymph node detection in pancreatic cancer Rev Esp Med Nucl Imagen Mol Schubert T Uphoff J Henke R P Wawroschek F Winter A Reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer Verification in consideration of the European guidelines BMC Urol “ Jaukovic L et al Lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions Hell J Nucl Med “ Subramanian S Pandey U Shah S Rangarajan V Samuel G An indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection Nucl Med Commun “ RuizDom­nguez J M IbarzServio L Garc­ade Manuel G Calaf Peris© O Intraoperative injection of 99mTcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery Actas Urol “ Schauer A J Specific developments in sentinel node labling using 99mTccolloids In The Sentinel Lymph Node Concept Springer Berlin Wang Y et al Gasphase chemistry of technetium carbonyl complexes Chem Phys “ O™Connor M K et al Comparison of Tc99m maraciclatide and Tc99m sestamibi molecular breast imaging in patients with Wang J Zhang R Evaluation of 99mTcMIBI in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer EJNMMI Res Br J Radiol Costa P et al Scintigraphic imaging with technetium99Mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats Acta Cir Bras “ Vera D R Wallace A M Hoh C K Mattrey R F A synthetic macromolecule for sentinel node detection 99mTcDTPAmannosyldextran J Nucl Med “ Hoh C K Wallace A M Vera D R Preclinical studies of [99mTc]DTPAmannosyldextran Nucl Med Biol “ Skuridin V et al Modified DTPA moleculebased nanocolloid radiopharmaceuticals J Radioanal Nucl Chem “ Filimonov V D et al Unusually stable versatile and pure arenediazonium tosylates Their preparation structures and synthetic applicability Lett “ Lukasz K Thin Layer Chromatography in Drug Analysis “ CRC Press London Skuridin V et al Radiopharmaceutical drug based on aluminum oxide Indian J Sci Technol 1017485 ijst2015v8i36 Sazonova S I et al Synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging Iran J Nucl Med “ Skuridin V S et al Synthesis and biological characterization of 99mTclabeled ciprofloxacin Pharm Chem J “ AcknowledgementsThis work was financially supported by Ministry of Education and Science of the Russian Federation RFMEFI57514X0034Author contributionsVS Conducting experimental research analysis and interpretation of the data Final approval for manuscript publication VS development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication EN development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication ES development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication AR conducting experimental research analysis and interpretation of the data Final approval for manuscript publication NV conducting experimental research analysis and interpretation of the data Final approval for manuscript publication RZ conducting tests of the functional suitability of drugs Preparation of the section Evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and Figures „– Final approval of the manuscript for publication of the manuscriptCompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to VSReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsScientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0c 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 license and indicate if changes 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 If material 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 obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c'
2
" vibrio cholerae are oxidasepositive bacteria that are classified into various serotypes based on the osurface antigen v cholerae serotypes are divided into two main groups the o1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v choleraenovc can cause choleralike diarrhea a pubmed search revealed that only cases of necrotizing fasciitis causedby novc have been recorded in the scientific literature to date we report the case of a japanese woman whodeveloped necrotizing fasciitis caused by novc after traveling to taiwan and returning to japancase presentation a 63yearold woman visited our hospital because she had experienced left knee pain for thepast days she had a history of colon cancer stage iv t3n3 m1a and had received chemotherapy she hadvisited taiwan days previously where she had received a massage she was diagnosed with septic shock owingto necrotizing fasciitis she underwent fasciotomy and received intensive care she recovered from the septic shockhowever after weeks she required an aboveknee amputation for necrosis and infection her condition improvedand she was discharged after weeks in the hospitals with the increase in tourism it is important for clinicians to check patients™ travel history cliniciansshould be alert to the possibility of necrotizing fasciitis in patients with risk factors necrotizing fasciitis caused bynovc is severe and requires early fasciotomy and debridement followed by intensive postoperative carekeywords necrotizing fasciitis vibrio cholerae taiwan massage septic shock polymyxin b correspondence kei610805gmailcom1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara 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 0ctsuruta bmc infectious diseases page of vibrio cholerae are curved gramnegative rod gnrbacteria that are oxidase positive they are classified intovarious serotypes based on the o surface antigen vcholerae serotypes are divided into two main groups theo1 and o139 group and the nono1nono139 group o1 and o139 v cholerae are related to cholera infection whereas nono1nono139 v cholerae novccan cause choleralike diarrhea novc are found as autochthonous microbes in coastal and marine environments outbreaks of choleralike illness caused bynovc have been reported in the united states o141and o75 former czechoslovakia o37 sudan o37peru o10 o12 and mexico o14 [“] moreovernovc can cause a range of extraintestinal infectionsincluding bacteremia meningitis pneumonia peritonitischolangitis salpingitis and softtissue infection seafood including oysters fishes shrimps clams musselsand apple snail is the most common source of infection a pubmed search revealed that only cases of necrotizing fasciitis caused by novc have beenreported in the scientific literature to date we reportthe case of a patient who developed necrotizing fasciitisand septic shock caused by novc which necessitatedan aboveknee amputation of her left legcase presentationa 63yearold woman visited minaminara general hospital in nara japan because she had experienced leftknee pain for days prior to her visit she had been diagnosed with colon cancer stage iv t3n3 m1a years and months previously and had undergone surgery and received chemotherapy her most recent doseof chemotherapy was administered days before herinitial consultation she had visited taiwan days previously where she had received a massage after themassage she developed gradually worsening pain in herlower left leg on presentation she was able to walkunaided and she reported her history of colon cancerand recent travel as we suspected that the pain in herleg could be due to necrotizing fasciitis we requestedmagnetic resonance imaging mri of her left lowerleg the images showed a swollen soleus muscle andposterior tibial muscle and the t2weighted imageshowed hyperintensity of the muscle tissue fig after the mri our patient™s condition deteriorated andthe following vital signs were observed blood pressurebp mmhg heart rate beatsmin respiratory rate breathsmin and temperature °cthe results of arterial blood gas analysis were as folˆ’ mmhglows ph paco2 mmhg hco3base excess be ˆ’ meql and lactate mmoll the patient™s laboratory test results were as followscreactive protein crp mgdl blood ureanitrogen bun mgdl creatinine mgdlprocalcitonin ngml nterminal probrain natriuretic peptide ntprobnp pgml and fibrinfibrinogen degradation products fdp μgmlinitiatedadministeredlowvenovenoushemodiafiltrationsurgery her blood pressure wasintravenous infusion of meropenem and noradrenaline wasand the patient underwentemergency surgery before the surgery the compartment pressure of her left leg was measured by simpleneedle manometry the pressures were as follows mmhg mmhg mmhg and mmhg in theanterior lateral superficial posterior and deep posterior compartments respectively some muscle tissuesin the anterior and deep posterior compartments werenecrotic for double incision fasciotomy a relaxationincision was made on her left knee and theaffected area was irrigated and debrided fig aftertheandtherefore wepolymyxin b directhemoperfusion pmxdhp to trap endotoxins andcontinuoususinghemofeel ch13 w toray medical co ltdurayasu japan as a slightly curved gnr that wasoxidase positive was detected in her blood we diagnosed her with necrotizing fasciitis and septic shockcaused by vibrio species we changed the antibioticsfrom meropenem to ceftriaxonelevofloxacin andminocycline we used the pmxdhp once again andtapered the dose of noradrenalin gradually wediscontinued noradrenalin on day postoperativelyon day postoperatively the anism was identifiedas novc theantibiotics wasconfirmed postoperatively on day and we discontinued levofloxacin table although the patient™sgeneral condition improved there was a discharge ofpus from the postoperative wound on day postoperatively a second debridement was performedseveral muscles in the patient™s left leg including theanterior tibial muscle had become necrotic and thenecrosis had spread to her knee on day postoperatively an aboveknee amputation was performedher vital signs and laboratory data obtained since admission are shown in fig her condition improvedand she was discharged weeks after admissionsusceptibility ofdiscussion and sixteen cases of necrotizing fasciitis caused by novchave been previously reported table [“] themajority of patients were exposed to seawater or hadan injury in rare cases vigorous massage is one ofthe risk factors of necrotizing fasciitis howeverthe patient in the present case had a risk of novcinfection because of colon cancer and immunosuppression due to chemotherapy as she received chemotherapy within a month thusthein this case 0ctsuruta bmc infectious diseases page of fig t2weighted magnetic resonance images of the patient™s lower legs a coronal image b axial image these images show that the soleusand posterior tibial muscles on the left lower leg indicated by red arrows are swollen and inflamedfig photographs of lesions in the patient™s leg the patient™s leg before surgery shows multiple large blisters 0ctsuruta bmc infectious diseases page of table susceptibility of antibioticsantibioticsampicillinminimal inhibitory concentrations piperacillinceftazidimeimipenemcilastatinamoxicillinclavulanategentamicinminocyclinechloramphenicolsulfamethoxazoletrimethoprimlevofloxacinfosfomycins s s ‰¦s s s ‰¦s ‰¦s ‰¦s ‰¦r the novc remains unknown assource ofthepatient did not report any exposure to sea water oreating seafood the only potential cause of injury toher left leg was the massage she received thereforewe speculate that the massage might have been thesource ofthe novc based on the circumstantialevidence we administered blood purification therapyusing pmxdhp and venovenous hemodiafiltrationfor septic shock although no previous studies havereported the use of pmxdhp for novc a studyreported the use of pmx for v vulnificus thirdgenerationtetracyclineandfluoroquinolone were used for severe vibrio infections tetracycline combined with the fluoroquinoloneorcephalosporinfollowed by oral fluoroquinolones or doxycycline wasrecommended for invasive novc infections [ ]an in vitro study revealed that cefotaxime and minocycline have a synergistic effect in the treatment forcephalosporinsaparenteralthirdgenerationfig change of vital signs and laboratory data during the hospital admission a changes in the patient™s vital signs during days “ ofhospitalization b changes in patient™s blood biochemistry during days “ of hospitalization atiii antithrombin iii crp creactive proteinfdp fibrinfibrinogen degradation products map mean arterial pressure nad noradrenaline 0ctsuruta bmc infectious diseases page of age sex underlying diseasestable clinical characteristics of patients with nono vibrio cholerae necrotizing fasciitisyear ofreportsourcesurgery amputation multiple debridementand antibiotics ticarcillinclavulanate imipenemgentamicin clindamycinrisk factors mdiabetes mellitussurvived usatreatmentoutcome country oantigen epidemiologicexposureexposure of achronic plantar ulcerto sand in abathhouse mcirrhosissurgery cefotaxime minocycline cefotaximesurvivedtaiwan f mcirrhosis congestiveheart failurecirrhosis diabetesmellitus mhepatitis csurgery ceftriaxonediedtaiwansurgical debridement ceftazidime doxycyclinediedtaiwan o56surgery antibiotics thirdgeneration cephalosporindoxycyclinediedtaiwanhandling seafood mhepatitis steroidssurgery antibiotics thirdgeneration cephalosporindoxycyclinesurvivedtaiwan mcirrhosissurgery clindamycin ceftazidime tetracyclinesurvivedtaiwansurgery antibioticssurgery antibioticsdiedtaiwandiedtaiwan m m mcirrhosis hepatitis cdiabetes mellituscirrhosis hepatitis bhepatitis c diabetesmellituscirrhosis diabetesmellitusexposure to seawaterprobable woundinfectionconsumption of rawseafoodseawater exposureinsect bite woundinfectionminor abrasionexposed to seawaterseawater exposuresurgery antibioticsdiedtaiwanseawater exposure mcirrhosissurgery antibioticssurvivedtaiwanseawater exposure mcopdsurgery antibioticssurvivedtaiwan mhiv hepatitis ccirrhosis mdiabetes mellitus michthyosis cellulitisnone mcopd chronic constructive pulmonary diseasesurgical debridement daptomycin levofloxacinsurviveditalyo137surgical debridement piperacillintazobactamfosfomycinsurgical debridement piperacillintazobactamtigecycline metronidazolesurgical debridementpenicillin gentamicin metronidazolesurvived austriadiedaustriaseawater exposuresurvived croatia o8seawater exposurev cholerae infections as patients with novcbacteremia require antibiotic treatment for at least month we administered ceftriaxone and minocycline for month necrotizing softtissue infectionscaused by novc are more lethal than those causedby v vulnificus to conclude we treated a woman with necrotizingfasciitis and septic shock caused by novc this caseillustrates that early fasciotomy and debridement arenecessary forsevere necrotizing fasciitis caused bynovc and prolonged intensive care may be requiredafter surgeryo139 vibrio cholerae ntprobnp nterminal probrain natriuretic peptidepmxdhp polymyxin b direct hemoperfusionacknowledgementsnoneauthors™ contributionskt tu tw kn and ku treated the patient kt tu hf reviewed the literatureand mainly wrote this report kn tw ku reviewed the literature andmodified this paper based on specialty orthopedics emergency departmentinfectious disease all authors have read and approved the manuscriptfundingnoneabbreviationsbun blood urea nitrogen fdp fibrinogen degradation productsgnr gramnegative rod mri magnetic resonance imaging novc nono1availability of data and materialsall data are included in this published 0ctsuruta bmc infectious diseases page of ethics approval and consent to participatethis case report was approved by the ethics review committee atminaminara general hospital and was conducted in accordance with thedeclaration of helsinki consent for participation is not applicableconsent 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 editor of this journalcompeting intereststhe authors declare that they have no competing interestsauthor details1emergency department minaminara general hospital ooazafukugamiooyodocho yoshinogun nara japan 2orthopedic departmentminaminara general hospital nara japan 3infectious diseases departmentminaminara general hospital nara japan 4department of emergency andcritical care medicine nara medical university nara japanreceived march accepted august referencesgardner ad venkatraman kv the antigens of the cholera group of vibriosj hyg lond “ hirk s huhulescu s allerberger f lepuschitz s rehak s weil s necrotizing fasciitis due to vibrio cholerae nono1nono139 after exposureto austrian bathing sites wien klin wochenschr “ dobrović k rudman f ottaviani d crnek sÅ¡ leoni f Å¡krlin j a rare case ofnecrotizing fasciitis caused by vibrio cholerae o8 in an immunocompetentpatient wien klin wochenschr “jain akc varma ak mangalanandan kh kumar h bal a surgical outcome ofnecrotizing fasciitis in diabetic lower limbs j diab foot comp “ikeda t kanehara s ohtani t furukawa f endotoxin shock due to vibriovulnificus infection eur j dermatol “su ba tang hj wang yy liu yc ko wc liu cy in vitro antimicrobialeffect of cefazolin and cefotaxime combined with minocycline against vibriocholerae nono1 nono139 j microbiol immunol infect “tsai yh huang tj hsu rww weng yj hsu wh huang kc necrotizing softtissue infections and primary sepsis caused by vibriovulnificus and vibrio cholerae nono1 j trauma “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations morris jg nono group vibrio cholerae a look at the epidemiology of anoccasional pathogen epidemiol rev “li m shimada t morris jg jr sulakvelidze a sozhamannan s evidence forthe emergence of nono1 and nono139 vibrio cholerae strains withpathogenic potential by exchange of oantigen biosynthesis regions infectimmun “dalsgaard a albert mj taylor dn shimada t meza r serichantalergs o characterization of vibrio cholerae nono1 serogroups obtained froman outbreak of diarrhea in lima peru j clin microbiol “isaacmárquez ap lezamadávila cm eslavacampos c navarroocaña acraviotoquintana a serotypes of vibrio cholerae nono1 isolated fromwater supplies for human consumption in campeche mexico and theirantibiotic susceptibility pattern mem inst oswaldo cruz “hughes jm hollis dg gangarosa ej weaver re noncholera vibrioinfections in the united states clinical epidemiologic and laboratoryfeatures ann intern med “deshayes s daurel c cattoir v parienti jj quilici ml de la blanchardière anono1 nono139 vibrio cholerae bacteraemia case report and literaturereview springerplus mubarak sj owen ca doubleincision fasciotomy of the leg fordecompression in compartment syndromes j bone joint surg am “ wagner pd evans sd dunlap j ballonlanda g necrotizing fasciitis andseptic shock caused by vibrio cholerae acquired in san diego californiawest j med “ko w chuang y huang g hsu sy infections due to nono1 vibrio choleraein southern taiwan predominance in cirrhotic patients clin infect dis “ cheng nc tsai jl kuo ys hsueh pr bacteremic necrotizing fasciitis causedby vibrio cholerae serogroup o56 in a patient with liver cirrhosis j formosmed assoc “tsai yh hsu rww huang kc chen ch cheng cc peng kt systemicvibrio infection presenting as necrotizing fasciitis and sepsis a series ofthirteen cases j bone joint surg am “ changchien ch bacteraemic necrotizing fasciitis with compartmentsyndrome caused by nono1 vibrio cholerae j plast reconstr aesthetic surg“ maraki s christidou a anastasaki m scoulica e nono1 nono139 vibriocholerae bacteremic skin and soft tissue infections infect dis lond “ ottaviani d leoni f rocchegiani e canonico c masini l pianetti a unusual case of necrotizing fasciitis caused by vibrio cholerae o137 j clinmicrobiol “ 0c"
0
Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earth™s species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleep™s quotidian œnormalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brain™s clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patient™s desired sleep time or augmenting factors that entrainthe body™s clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require “ h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon œsiesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period œCatchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances œmorning larks latephase delays œnight owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of “ weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ‰¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airway™s soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisorders”specifically sleep walking”also occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women “ in males and “ in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [“] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economo™s encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while œasleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of “ OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in œthe loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleep”or its encephalopathic analog”may affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i
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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 ˆ—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 anti‚ammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these find 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 benefit 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 find out a new se ries of nep inhibitors to the best of our knowledge this is the first 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 ˚a 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 refine review and modify and minimize processes in protein preparation wizard missing side chains and residues were filled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond ˚a 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 field and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workflow 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 field 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 affinities 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 file 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 field to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme profile 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 coefficient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of five were calculated induced fit 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 inducedfit 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 ifd“sp 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 refinement of all residues within ˚a 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 inducedfit protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula ˆ— prime_energy ˆ— glide score ˆ—ifd score glide_ecoul molecular dynamics md simulation the flexibility 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 fined spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol ˚a balanced at k tem perature and bar pressure via npt ensemble in the final 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 profile 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 workflow was used and the cubic box of specific 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 final torsional refinement 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 filtered 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 identified 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 finding indicates that the selected drugs may act as nep inhibitors induced fit 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 fit docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the flexible 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 first 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 confirmers 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 profile was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of five given in table all the selected drugs obey the lip inski rule of five 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 flexibility which is insufficient 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 fit 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 ˚a and ˚a respectively the rmsd values were found to be in the acceptable range ˚a 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 ˚a and ˚a for protein and ligand respectively for complex the rmsd values were found to be ˚a 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 ˚a 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 signifi 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 five 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 anti‚ammatory 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 affinity 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 first 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 drug™s 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 identified 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
Primary squamous cell carcinoma ofis anextremely rare aggressive malignancy with a poor prognosis However almost noreportthus far has investigated the microvasculature of ThyPSCC imaged usingcontrastenhanced ultrasoundthe thyroid ThyPSCCCase Report A 59yearold male patient presented to our hospital with progressivelyworsening hoarse voice symptoms for days and was diagnosed with left unilateralvocal fold palsy Ultrasonography revealed a solitary marked hypoechoic thyroid nodulewith an unclear boundary in the inferior part of the left lobe Color Doppler flow imagingshowed a poor blood flow signalinside this nodule Contrastenhanced ultrasoundimages showed a persistent low peak enhancement of the nodule from its periphery to itscenter The timeintensity curve displayed a washin time of s a time to peak of s apeak signal intensity of and a washout time of s for the thyroid tumor Finallyleft hemithyroidectomy of the thyroid tumor was performed and histopathologic andimmunohistochemical evaluations confirmed the diagnosis of ThyPSCC Postoperativelythe patient received a combination therapy of chemotherapy radiotherapy and targetedtherapy but the patient died months after surgeryPrimary squamous cell carcinoma ofConclusionthe thyroid is a rare butaggressive malignancy of the thyroid Herein we reported a case of ThyPSCC and itsultrasonography and pathologic findingsKeywords thyroid cancer thyroid nodules TNs thyroid ultrasound US primary squamous cell carcinomacontrast enhanced ultrasound CEUSINTRODUCTIONPrimary squamous cell carcinoma of the thyroid ThyPSCC is a rare thyroid malignancy withhigh aggressiveness and poor prognosis comprising ˆ¼“ of all primary thyroid carcinomas“ Owing to the rapidly progressing and highly invasive nature of the malignancy patients withThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage becauseof its rare incidence and lack of typical imaging findings Thyroid ultrasonography and fineneedle aspiration biopsy FNAB are the diagnostic tools ofchoice for evaluating patients with suspected thyroid nodules Contrastenhanced ultrasoundCEUS as a relatively novel US technique is used to investigate the microvasculature of thyroidnodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of ThyroidEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byPasqualino MalandrinoUniversity of Catania ItalyDaniela PasqualiUniversity of Campania LuigiVanvitelli ItalyCorrespondenceChengcheng NiuniuchengchengcsueducnSpecialty sectionThis was submitted toCancer Endocrinologya section of the journalFrontiers in EndocrinologyReceived February Accepted June Published August CitationChen S Peng Q Zhang Q and Niu C ContrastEnhanced Ultrasoundof Primary Squamous Cell Carcinomaof the Thyroid A Case ReportFront Endocrinol 103389fendo202000512Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaImaging Reporting and Data Systems for ultrasonographicfeatures “ However very few published studies havereported the use of ultrasonography for ThyPSCC To ourknowledge this is the first case describing the CEUS findingsof ThyPSCCreached its peak [time to peak TTP] at s with a peakintensity of Then the nodule slowly declined until allthe microbubbles washed out at s Figures 1CD Based onits malignant conventional ultrasound features and the poormicrovasculature revealed by CEUS we inferred that the nodulewas a malignant tumorCASE REPORTA 59yearold male patient presented to our hospital withprogressively worsening hoarse voice symptoms for daysand was diagnosed with left unilateral vocal fold palsy Ahighresolution ultrasound instrument Siemens Acuson S3000Mountain View CA USA equipped with a to 9MHz linearprobe was used Thyroid ultrasonography revealed a solitary — — 26cm3 marked hypoechoic thyroid nodule with anunclear boundary in the inferior part of the left lobe AThis nodule exhibited many malignant ultrasound featuressuch as solid components hypoechogenicity and microlobulatedmargins Color Doppler flow imaging CDFI showed poorblood flow signals in the nodule B Contrastenhancedultrasound was performed with a bolus intravenous injectionof mL of SonoVue Bracco Milan Italy followed by mLof saline Contrast pulse sequencing technology was used andthe timeintensity curves TICs of the nodule were calculatedThe nodule began to be slowly enhanced from the peripheryto the center at s washin time and the enhancementAfterneckthepositronultrasonographyemissiontomography“computed tomography was carried for evaluatingthesituation of distant metastases Positron emissiontomography“computed tomography showed a mass withincreased glucose metabolism in the inferior part of the leftthyroid lobe A which indicated it as a malignantmass whereas there was no evidence of lymph nodes metastasisand distant metastases Then ultrasonographyguided FNABwas performed for the left thyroid mass immediately Cytologicexamination by fineneedle aspiration FNA revealed sheets oftumor cells with giant deepstained nuclei Bethesda categoryV B Finally a left hemithyroidectomy of the thyroidtumor was undertaken The lower edge of the tumor reachedthe upper mediastinum and the depth of the tumor invadedthe esophagus and trachea which could not be completelyremoved According to the eighth edition of the AmericanJoint Committee on CancerTumor Lymph Node MetastasisTNM staging system the patient was in TNM stage IIIT4a N0 M0 Histopathological examination of hematoxylinand eosin staining showed that a carcinoma in the inferiorFIGURE Ultrasonography images of primary squamous cell carcinoma of the thyroid A Longitudinal grayscale sonography revealed a solid marked hypoechoicthyroid nodule in the inferior part of the left lobe B Color Doppler flow imaging showed a poor blood flow signal inside this nodule C Contrastenhanced ultrasoundimage showed a persistent low peak enhancement of the nodule at s D Timeintensity curves displayed the washin time of s TTP of s peak signalintensity of and washout time of s for the thyroid tumorFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE A A positron emission tomography“computed tomography scan showed increased 18Ffluorodeoxyglucose metabolism in the left neck mass BPreoperative fineneedle aspiration cytology of the mass demonstrated a few sheets of malignantlooking tumor cells with giant deep stained nuclei hematoxylin andeosin magnification — FIGURE Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid A magnification — B magnification — C magnification — D magnification — part of the thyroid lobe A had no obvious palisadearrangementintercellular bridges or keratinization with acancer pearl Figures 3B“D Immunohistochemically tumorcells were positive for cytokeratin CK19 Acytokeratin and CK56 B epithelial membraneantigen EMA C p40 D p63 Aand Ki67 B and negative for thyroglobulinTG C and thyroid transcription factor TTF1D In view of these findings the tumor was diagnosedas poorly diï¬erentiated ThyPSCC Postoperatively the patientreceived two cycles of chemotherapy with docetaxelcisplatinintensitymodulated radiotherapy and nimotuzumabtargetedtherapy However the patient died months after surgeryDISCUSSIONPrimary squamous cell carcinoma of the thyroid is a thyroidmalignancy with extremely rare incidence and the clinicaldiagnosis and treatment guidelines for this disease have noconsensus The biological behavior of ThyPSCC is aggressiveFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell CarcinomaFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A CK19 BCK56 C EMA D p40 all of which were deeply stained positiveFIGURE Immunohistochemical staining of primary squamous cell carcinoma of the thyroid magnification — Immunohistochemical staining for A p63 B Ki C TG D TTF1 and p63 was deeply stain positive Ki67 proliferation index was TG and TTF1 did not stain negativeFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alThyroid Primary Squamous Cell Carcinomaand the prognosis is poor with a median overall survival of “ months which depends on the diï¬erent tumor grades Yang et al using the Surveillance Epidemiology and End ResultsProgram database reported that poorly diï¬erentiated tumrade occupied the highest percentages of all graded tumors andthe median survival was months which is similar to the survivaltime in our case Highfrequency ultrasound as the basic imaging modality inthe diagnosis of thyroid nodules has found gradually increasingdiï¬erentiated thyroid cancers over recent years Theultrasonography imaging findings of ThyPSCC have seldombeen published Regarding the ultrasonography findings Chenet al reported that ThyPSCC presented as a thyroid masswith eggshell calcification peripheral soft tissue with a blurredmargin and minimal vascular signals on CDFI sonographyIn the case of Jang et al ThyPSCC presented as a largewelldefined lobulated heterogeneously hypoechoic mass withdiï¬use microcalcifications on ultrasonography Kondo et al reported that a welldiï¬erentiated ThyPSCC showed acystic hypoechoic mass with a smooth margin and rapidlygrew with margin change blurring in year In our case thispoorly diï¬erentiated ThyPSCC presented as a solitary markedhypoechoic thyroid mass with an irregular margin and unclearboundary with a normal thyroid The irregular margin andunclear boundary with normal thyroid corresponded to tumorinvasion with adjacent tissue infiltration which is consistentwith the findings during the operation that tumor invasion withthe esophagus cannot be completely removed Poor blood flowsignals on CDFI sonography and persistent hypoenhancement onCEUS of the mass are consistent with squamous cell carcinomawhich has no obvious vascularity on pathologic examinationMany studies have investigated the application of CEUS toimprove the diagnostic accuracy of thyroid nodules despiteits usage in ThyPSCC being scarce Zhang et al foundthat highcircularequal enhancement indicated benign thyroidnodules and low enhancement indicated malignant thyroidnodules Ma et al investigated whether incomplete noring or heterogeneous enhancement later washin time andlow peak intensity on CEUS were independent risk factorsin predicting malignantthyroid nodules Deng et al detected that papillary thyroid carcinomas PTCs exhibited lowenhancement a lower peak signal intensity and a lower areaunder the curve AUC than peripheral thyroid parenchyma onCEUS In our study the TICs of CEUS for ThyPSCCshowed a washin time of s a TTP of s a peak signalintensity as low as and a washout time of s Thisis similar to the results of PTCs with a slow washin time alower peak signal intensity and a lower AUC as in previousreports To our knowledge no reports on CEUS imagingfindings of ThyPSCC have appeared in the Englishlanguageliterature According to Jang et al ThyPSCC showed a largeheterogeneously enhancing thyroid mass with a large centralnonenhancing portion on enhanced CT which correspondedwell with the squamous cell carcinoma portion with a necroticportion in pathologic staining Because of the rapid growth ofsquamous tumor cells relatively few interstitial blood vessels intumors were related to the low peak signal intensity and low AUCon CEUSisusefulstainingWith increasing malignancy in squamous cell carcinoma thetypical squamous cell carcinoma findings of intercellular bridgesand keratinized cancer pearl can decrease or disappearImmunohistochemicalin diagnosingprimary thyroid cancer In this case positivity for CK56and EMA and negativity for TTF1 and TG expressionpredicted squamous cell carcinoma derivation and excludedthe possibility ofthese common tumors Furtherpositivity for p63 and Ki67 expression as poor prognosticmarkers was associated with its poorly diï¬erentiated tumrade CONCLUSIONPrimary squamous cell carcinoma of the thyroid is an extremelyrare tumor and very few studies describe its ultrasonographicimaging findings It is difficult to establish a clinical guidelinefor diagnosis Our case presents the CEUS features of ThyPSCCindicating that the TICs of ThyPSCC are similar to the enhancingparameters of PTCs with a slow washin time a lower peak signalintensity and a lower AUCDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studies involving human participants were reviewed andapproved by the Ethics Committee of Second Xiangya HospitalCentral South University China The patientsparticipantsprovided their written informed consentto participate inthis study Written informed consent was obtained from theindividuals for the publication of any potentially identifiableimages or data included in this AUTHOR CONTRIBUTIONSAll authors listed have made a substantial direct and intellectualcontribution to the work and approved it for publicationFUNDINGof ChinaThis project was funded by the National Natural ScienceFoundationProvincialNatural Science Foundation of China 2018JJ2575 andHunan Provincial Health Commission Research FoundationProject B2019166 HunanFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cChen et alREFERENCES Yang S Li C Shi X Ma B Xu W Jiang H et al Primary squamous cellcarcinoma in the thyroid gland a populationbased analysis using the SEERdatabase World J Surg “ 101007s00268019049062 Limberg J Ullmann TM Stefanova D Finnerty BM Beninato T Fahey TJet al Prognostic characteristics of primary squamous cell carcinoma of thethyroid a national cancer database analysis World J Surg “ 101007s00268019050985Thyroid Primary Squamous Cell Carcinomacontrastenhanced ultrasound Ultrasound Med Biol “ 1016jultrasmedbio201810020 Casella C Ministrini S Galani A Mastriale F Cappelli C Portolani NThe new TNM staging system for thyroid cancer and the risk of diseasedownstaging Front Endocrinol 103389fendo201800541 Zhang Y Zhou P Tian SM Zhao YF Li JL Li L Usefulness of combineduse of contrastenhanced ultrasound and TIRADS classification for thediï¬erentiation of benign from malignant lesions of thyroid nodules EurRadiol “ 101007s003300164508y Koyama S Fujiwara K Nosaka K Fukuhara T Morisaki T MiyakeN et al Immunohistochemical features of primary pure squamous cellcarcinoma in the thyroid an autopsy case Case Rep Oncol “ Zhang YZ Xu T Gong HY Li CY Ye XH Lin HJ et al Application ofhighresolution ultrasound realtime elastography and contrastenhancedultrasound in diï¬erentiating solid thyroid nodules Medicine95e5329 101097MD00000000000053290000579220161108000016 Wang SS Ye DX Wang B Xie C The expressions of keratins andP63 in primary squamous cell carcinoma ofthe thyroid gland anapplication of raman spectroscopy Onco Targets Ther “ 102147OTTS229436 Chen CY Tseng HS Lee CH Chan PW Primary squamous cellcarcinoma of the thyroid gland with eggshell calcification sonographicand computed tomographic findings J Ultrasound Med “ 107863jum201029111667 Yasumatsu R Sato M Uchi R Nakano T Hashimoto K Kogo R et al Thetreatment and outcome analysis of primary squamous cell carcinoma of thethyroid Auris Nasus Larynx “ 101016janl201707009 Kao NH Tan CS H Koh AJ The utility of immunohistochemistry indiï¬erentiating metastatic primary squamous cell carcinoma of the thyroidfrom a primary lung squamous cell carcinoma Case Rep Endocrinol “ Jang JY Kwon KW Kim SW Youn I Primary squamouscarcinoma ofandtomographic“ 1014366usg13022cellrecurrence ultrasonographicthyroid gland with localUltrasonographycomputedfindings Raggio B Barrcarcinomacell 1031486toj180002J Ghandour Z Friedlander P Primary squamousof“thyroid OchsnertheJ Kondo T Matsuyoshi A Matsuyoshi H Goto R Ono K Honda Y et alA case of primary thyroid squamous cell cancer transformation frombenign tumour associated with chronic thyroiditis BMJ Case Rep 2009bcr1020081137 101136bcr1020081137 Ma JJ Ding H Xu BH Xu C Song LJ Huang BJ et al Diagnosticand contrastmalignant101089thy201performances ofenhancedultrasonographythyroid nodules Thyroid“ color dopplergrayscalepredictingfindingsvariousin Deng J Zhou P Tian SM Zhang L Liofefficacydiagnosticofradiationdiï¬erentiating9e90674 101371journalpone0090674PONED1330329imagingthyroidandnodulescontrastenhancedimpulseforcesolidfocalJL Qian Y ComparisonacousticinuseultrasoundcombinedPLoS ONEtheir Haugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEet al American thyroid association management guidelines for adultpatients with thyroid nodules and diï¬erentiated thyroid cancer the Americanthyroid association guidelines task force on thyroid nodules and diï¬erentiatedthyroid cancer Thyroid “ 101089thy20150020 Tessler FN Middleton WD Grant EG Hoang JK Berland LL Teefey SAet al ACR thyroid imaging reporting and data system TIRADS whitepaper of the ACR TIRADS committee J Am Coll Radiol “ 101016jjacr201701046 Kwak JY Han KH Yoon JH Moon HJ Son EJ Park SH et al Thyroidimaging reporting and data system for US features of nodules a step inestablishing better stratification of cancer risk Radiology “ 101148radiol11110206radiol11110206 Peng Q Niu C Zhang Q Zhang M Chen S Mummified thyroid nodulesconventional and contrastenhanced ultrasound features J Ultrasound Med “ 101002jum14712 Peng Q Niu C Zhang M Chen S Sonographic characteristics ofpapillary thyroid carcinoma with coexistent hashimoto™sthyroiditisconventional ultrasound acoustic radiation force impulse imaging and Struller F Senne M Falch C Kirschniak A Konigsrainer A Mullerthe thyroid case report andS Primary squamous cell carcinoma ofsystematic review of the literature Int J Surg Case Rep “ 101016jijscr201706011 Wang W Ouyang Q Meng C Jing L Li X Treatment optimization andprognostic considerations for primary squamous cell carcinoma of thethyroid Gland Surg “ 1021037gs20191107Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Chen Peng Zhang and Niu This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'
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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 People™sHospital 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 ThirdPeople™s 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 strategy3“Comparisons 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 specific 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 patient™sthreatdistinction 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 Affiliated 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 availablethroughoutidentifiedfrom the orthopaedics department of theFirst Affiliated 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 defined as partial or completereplacement of the prosthesis12 Looseningof the acetabulum andor stem componentstomographycompared usingas well as dislocation were defined based ona previous description13 Periprostheticconfirmed 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 classification and major orthopaedic complications were compared using the chisquaretest or the Mann“Whitney test A Kaplan“Meier survival curve was used to assess theprobability of revision Hazard ratios werecalculated using a Cox proportional hazards model The significance 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 patients™baselinesimilarbetween 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 final analysis p CTA 06 prior to surgery vs 06 at final 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 “ monthsSignificant 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 confidence“p¼ 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 Kaplan“Meier 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 findings 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 efficacyof 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 Kaplan“Meiersurvival 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 materialinfluencesthepeak 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 surgeon™s 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 insufficient 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 theKaplan“Meier 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 findings 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 findings may beconducive to alleviating continuing debateregarding which prosthesis UTA or CTAis more suitable for the elderly populationA future prospective study may be essentialto confirm whether our conclusion continues to be acceptable as the followup timeincreasesDeclaration of conflicting interestThe authors declare that there is no conflict ofinterest 0cFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit 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 S232“S237 Hanly RJ Whitehouse SL Lorimer MFet al The outcome of cemented acetabularcomponents in total hip arthroplasty forosteoarthritis defines a proficiency 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 influence 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 fixations 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 five 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
"were a fraction of those needed for the photon plans (). This translates to a beam on time per field of between 5 and 10 seconds for the PT-SABR plans compared to 75 to 90 seconds for photon plan. This 5 to 10 second time estimate is based on a conservative 1 nC/sec dose rate however new proton centers may be able to achieve greater than 2 nC/sec thereby reducing this time by a factor of 2. By decreasing the treatment time to less than 10 seconds per field breath-hold techniques may be better tolerated in greater number of lung cancer patients with suboptimal lung function. Breath-hold technique would minimize tumor motion (i.e. ITV) leading to a smaller overall irradiation volume and interplay would not be a significant issue [8]. Spot scanning proton therapy that utilizes the Bragg peak would require a larger planning volume due to the various uncertainties that need to be taken into account; and it would require a longer treatment time due to the use of multiple proton energies. Each change in energy requires several seconds (2 to 7) and at least 5 to 10 energies would be required for these treatments. Volumetric modulated arc therapy (VMAT) with photons may decrease treatment times compared to multiple static-gantry beams. However VMAT comes at the cost of larger volumes of normal tissue receiving low doses of radiation since the beam is continuously on as it rotates about the patient. The use of four to five proton transmission beams achieves both shorter treatment times as well as a lower integral dose to the body. The dosimetric data of the normal tissues in the photons plans met the constraints of RTOG 0915. The dosimetric gains of protons over these plans may be considered modest and the statistical analysis comparing plans is limited by the small sample size. However in some plans the dose to particular critical ans can be avoided completely without compromising target coverage by choosing beam arrangements appropriately. This may be beneficial in treating patients with tumors in challenging locations [9] or recurrent tumors that have had prior radiotherapy. The interim analysis of RTOG 0617 reported local failure rates of 25% and 34% in the standard and high dose RT arms [10] and therefore re-irradiation may play a role in this subset of patients who fail after definitive chemoradiotherapy. For these patients keeping dose at or near zero to the spinal cord heart lungs or other critical structures is feasible with protons. Planning with PT-SABR using only transmission beams without the Bragg peak is feasible. This proof of principle as described in our study eliminates the uncertainty of proton dose distribution in lung tumors which has the potential to underdose the target and overdose surrounding normal tissues. Proton therapy planning with this technique also demonstrates better sparing of normal tissues and fast treatment times than photon plans. Further study of this novel approach to proton SABR is warranted. The authors thank Katy Nelson for maintaining the SABR database. References 1 GeD HillbrandM StockM DieckmannK PotterR (2008) Can protons improve SBRT for lung lesions? Dosimetric considerations. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology88: 368“37518405986 2 HoppeBS HuhS FlampouriS NicholsRC OliverKR et al (2010) Double-scattered proton-based stereotactic body radiotherapy for stage I lung cancer: a dosimetric comparison with photon-based stereotactic body radiotherapy. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology97: 425“43020934768 3 MacdonaldOK KruseJJ MillerJM GarcesYI BrownPD et al (2009) Proton beam radiotherapy versus three-dimensional conformal stereotactic body radiotherapy in primary peripheral early-stage non-small-cell lung carcinoma: a comparative dosimetric analysis. International journal of radiation oncology biology physics75: 950“958 4 WestoverKD SecoJ AdamsJA LanutiM ChoiNC et al (2012) Proton SBRT for medically inoperable stage I NSCLC. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer7: 1021“1025 5 PaganettiH (2012) Range uncertainties in proton therapy and the role of Monte Carlo simulations. Physics in medicine and biology57: R99“11722571913 6 SecoJ PanahandehHR WestoverK AdamsJ WillersH (2012) Treatment of non-small cell lung cancer patients with proton beam-based stereotactic body radiotherapy: dosimetric comparison with photon plans highlights importance of range uncertainty. International journal of radiation oncology biology physics83: 354“361 7 VideticGM HuC SinghA ChangJY ParkerW et al (2013) Radiation Therapy Oncology Group (RTOG) Protocol 0915: A Randomized Phase 2 Study Comparing 2 Stereotactic Body Radiation Therapy (SBRT) Schedules for Medically Inoperable Patients With Stage I Peripheral Non-Small Cell Lung Cancer. International journal of radiation oncology biology physics87: S3 8 KeallPJ MagerasGS BalterJM EmeryRS ForsterKM et al (2006) The management of respiratory motion in radiation oncology report of AAPM Task Group 76. Medical physics33: 3874“390017089851 9 RegisterSP ZhangX MohanR ChangJY (2011) Proton stereotactic body radiation therapy for clinically challenging cases of centrally and superiorly located stage I non-small-cell lung cancer. International journal of radiation oncology biology physics80: 1015“1022 10 BradleyJD PaulusR KomakiR MastersGA ForsterK et al (2013) A randomized phase III comparison of standard-dose (60 Gy) versus high-dose (74 Gy) conformal chemoradiotherapy with or without cetuximab for stage III non-small cell lung cancer: Results on radiation dose in RTOG 0617. Journal of Clinical Oncology31: 7501 Cancer Cancer cncr Cancer 0008-543X 1097-0142 BlackWell Publishing Ltd Oxford UK 24752945 4140446 10.1002/cncr.28714 Original s A phase 2 cooperative group adjuvant trial using a biomarker-based decision algorithm in patients with stage I non-small cell lung cancer (SWOG-0720 NCT00792701) Bepler Gerold MD PhD 1 Zinner Ralph G MD 2 Moon James MS 3 Calhoun Royce MD 4 Kernstine Kemp MD 5 Williams Charles C MD 6 Mack Philip C PhD 4 Oliveira Vasco PhD 1 Zheng Zhong MD PhD 6 Stella Philip J MD 7 Redman Mary W PhD 2 Gandara David R MD 4 1 Karmanos Cancer Institute Detroit Michigan 2 The University of Texas MD Anderson Cancer Center Houston Texas 3 SWOG Statistical Center Seattle Washington 4 University of California at Davis Sacramento California 5 City of Hope Duarte California 6 H. Lee Moffitt Cancer Center Tampa Florida 7 Michigan Cancer Research Consortium Community Clinical Oncology Program Ann Arbor Michigan Corresponding author: Gerold Bepler MD PhD Karmanos Cancer Institute 4100 John R Detroit MI 48201; Fax: (313) 576-8628; beplergkarmanos. 01 8 2014 18 4 2014 120 15 2343 2351 10 2 2014 17 3 2014 18 3 2014 2014 The Authors. Cancer published by Wiley Periodicals Inc. on behalf of American Cancer Society 2014 This is an open access under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License which permits use and distribution in any medium provided the original work is properly cited the use is non-commercial and no modifications or adaptations are made. BACKGROUND This cooperative group adjuvant phase 2 trial in patients with completely resected stage I non-small cell lung cancer with tumor diameters measuring ??2 cm was designed to assess the feasibility and preliminary efficacy of assigning patients to therapy or observation using a molecularly based decision algorithm. METHODS At least a lobectomy and sampling of recommended mediastinal lymph node stations good Zubrod performance status adequate an function and a formalin-fixed and paraffin-embedded tumor specimen were required. Excision repair cross-complementing group 1 (ERCC1) and ribonucleotide reductase M1 (RRM1) were analyzed using immunofluorescence-based in situ automated quantitative image analysis and categorized as high or low using prespecified cutoff values. Patients with high ERCC1 and RRM1 were assigned to observation and all others to 4 cycles of cisplatin and gemcitabine. Feasibility was defined as treatment assignment within 84 days from surgery in >?85% of patients. Secondary objectives were to estimate the 2-year survival. RESULTS Treatment assignment met the feasibility criteria in 88% of eligible patients (71 of 81 patients). The collective 2-year disease-free and overall survival rates were 80% and 96% respectively. Protein levels for RRM1 fell within the previously established range ERCC1 levels were slightly lower than expected and they were significantly correlated (correlation coefficient 0.4). "
1
Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedObjective To investigate whether preventive administration of a proton pump inhibitor PPI can reduce the occurrence anddevelopment of traumatic granuloma TG following type IVVI cordectomy Methods We retrospectively analyzed the statusof postoperative granulomas in patients who underwent type IVVI cordectomy due to glottic cancer and determinedwhether postoperative administration of a PPI had any impact on granuloma formation and development Results Thepercentage and number of patients with granuloma in the PPI treatment group experimental group at the 1st 2nd 3rd and6th month following surgery were and respectively The percentageand number of patients with granuloma in the noPPI group control group were and respectively The granuloma percentage of the PPI treatment group was lower than that of thecontrol group at all postoperative time points assessed The diï¬erences were not statistically significant at the 1st monthp but were statistically significant at the 2nd and 3rd months after surgery p p ConclusionPreventive use of a PPI in patients after type IVVI cordectomy can shorten the TG recovery duration and may reduce theseverity of TG but it cannot prevent TG from occurring Our results should be confirmed by prospective randomized controlledtrials with large sample sizes IntroductionLaryngeal squamous cell carcinoma LSCC is a commonhead and neck cancer It had an incidence of approximately in China between and [] and new cases were reported worldwide in [] Itis estimated that there will be new cases worldwidein [] Approximately twothirds of LSCCs originatein the glottic area The presence of hoarseness in patientswith earlystage glottic cancer GC prompts the patients toseek medical treatment Anatomically the larynx is surrounded by cartilage and has sparse lymphatic tissue As aresult patients with GC are mostly diagnosed at an earlystage which is clinically defined as T12N0M0 []In recent years surgery has been gradually abandoned in the treatment of earlystage GC and has beenreplaced with transoral microsurgery TM or radiotherapyTM has the advantages of being minimally invasive and having a high laryngeal preservation rate and high costeï¬ectiveness Transoral laser microsurgery TLM is the mostcommon surgical method used for GC although a few studies in the literature have adopted transoral coblation microsurgery TCM Although TM methods diï¬erthermalinstruments are often needed for the surgery [“]Traumatic granuloma TG is a common complication ofTM especially after type IVV cordectomy Mild TG mayonly manifest as hoarseness foreign body sensation and frequent throat clearing The severe granuloma may cause or becomplicated by perichondritis which leads to severe symptoms such as dyspnea Additionally this granulation isbelieved to be an important factor in the formation of glotticweb and larynx stenosis [“] Reflux is an important 0cBioMed Research Internationalfactor that aï¬ects granuloma formation Proton pump inhibitors PPI are often used empirically in the treatment ofpatients with postoperative granulomas [ ]We found in our previous clinical practice that patientswith a wide range of resections such as type IVVI cordectomies have a higher risk of postoperative granuloma Thiscan sometimes be very severe even requiring temporary tracheotomy to alleviate dyspnea which greatly and adverselyimpacts patients™ quality of life PPIs have been empiricallyused for the treatment of patients with postoperative granuloma and evidence of its eï¬ectiveness has been published[] However there has not been any research on the prevention of postoperative granuloma formation with PPIsTherefore we initiated PPI treatment for patients who hadundergone type IVVI cordectomies and compared theresults with those of patients who had also undergone thistype of surgery but were not treated with PPIs to evaluatethe eï¬ect of PPI treatment for preventing postoperative granuloma formation in this patient population Materials and Methods Patients This was a retrospective study Patient inclusioncriteria i patients with vocal cord cancer who agreed toundergo type IV V and VI cordectomies ii patients whodid not undergo preoperative and postoperative radiotherapy Exclusion criteria i patients who were complicatedwith diabetes and were not treated routinely ii patientswho used PPIs regularly iii patients who continued tosmoke and drink after surgeryA total of patients between January and December were recruited as the control group who did not use PPIsimmediately after surgery and did not take PPIs persistentlyA total of patients between January and June were recruited as the experimental group PPI treatment group who took PPIs immediately after the surgeryand routinely Since this was a retrospective study only thepatients in the experimental group underwent preoperativereflux symptom index RSI and reflux finding score RFSassessments Patients with an RSI score points andoran RFS ‰¥ points were considered to have laryngopharyngeal reflux disease LPRD [ ]All patients signed an informed consent form prior to thesurgery All clinical experiments conformed to the guidelinesissued by the committee on clinical research of Peking UnionMedical College Hospital PUMCH Ethics Committeeapproval was obtained at PUMCH and all patients providedspecific written informed consent Surgical Procedure According to the European Laryngological Society classification endoscopic cordectomies includethe following types type IV total cordectomy type Vaextended cordectomy encompassing the contralateral cordtype Vb extended cordectomy encompassing the arytenoidtype Vc extended cordectomy encompassing the ventricularband type Vd extended cordectomies encompassing thesubglottis and type VI extended cordectomies encompassing the anterior commissure [ ] During the surgerythe larynx was fully exposed with a selfretaining laryngo°scope Karl Storz Tuttlingen Germany and the tumor°was resected en bloc under direct visualization of a orlaryngoscope Karl Storz Tuttlingen Germany using amodel coblator ArthroCare Corp Sunnyvale CA witha coblation level of and a coagulation level of Postoperative Treatment Procedure and FollowUp Thepatients in the experimental group were treated with intravenous omeprazole mg per day before the recovery of oralfeeding cases recovered oral feeding on the first day aftersurgery and cases recovered oral feeding on the third dayThen they were given mg oral omeprazole twice daily minutes before breakfast and dinner for consecutiveweeks The patients in the control group were not treatedwith PPIs but if severe granulomas formed during followup and required intervention they were also given PPIs routinely patients in the control group developedgranulomas But only patients developed severe granulomas on the 2nd 3rd and 3rd month after surgery respectively and began to be given PPIs for weeks the same asthe experimental group while the other cases in the control group were not given PPIs throughoutthe wholefollowup period All patients were treated with cefuroximeat mg twice daily for week to prevent infection Afterthe surgery the patients were required to quit smoking anddrinking and engage in reasonable vocal useThe followup procedure included regular checkups at and months after surgery If a granuloma was detectedduring the 3month checkup the patients were followedmonthly until the granuloma disappeared Postoperativegranuloma was defined as relatively smooth tissue in the surgery region that protruded from the surrounding area thatmay be attached to a pseudomembrane The granulomaand the surrounding area did not have obvious vascularhyperplasia The proportion of patients with postoperativegranuloma was used as an observation indicator Additionally the number of unscheduled visits and the rate of reoperationincluding tracheotomy and granulectomy wererecorded as indicators of granuloma severityDuring followup if the granuloma was found to severelyimpact vocalization andor breathing or if patients were suspected of having a recurrent tumor the granuloma was surgically resected and the specimen was sent for examination Statistical Analysis Data were statistically analyzed withSPSS software SPSS Chicago IL Nonnormally distributed quantitative data are represented as median and interquartile range and were subjected to the Wilcoxon ranksumtest Normally distributed measurement data are representedas mean ± standard deviation and were subjected to theindependent sample t test Count data were subjected to thechisquared test Diï¬erences with p were consideredstatistically significant Results Baseline Characteristics The baseline data of the patientsin the PPI treatment group and the control group are shownin Table Among the patients in the PPI treatment 0cBioMed Research InternationalTable General information DiscussionVariableSex MFAge yearsTumor staging T1aT1bT2Surgery type IVVVIPPIn ± Controln ± p valuegroup patients were male and patient was female withan average age of ± years patients were at theT1 stage and patients were at the T2 stage patients underwent type IV surgery patients underwent type V surgeryand patients underwent type VI surgery Among the patients in the control group patients were male and patient was female with an average age of ± years patients were at the T1 stage and patients were at theT2 stage patients underwent type IV surgery patientsunderwent type V surgery and patients underwent typeVI surgery The two groups of patients did not diï¬er significantly in the baseline conditions Table Postoperative Granulation The numbers and percentageof patients with granuloma in the PPI treatment group atthe 1st 2nd 3rd and 6thmonth followup were and respectively The numbers and percentage of patients with granuloma in the control group at those time points were and respectively Although the percentage of granuloma in the PPItreatment group was lower than that of the control group ateach stage only the diï¬erences at the 2nd and 3rd monthsafter surgery were statistically significant p and p respectively Only one patient in the PPItreatment group required a second surgery due to persistentgranulation patients in the control group underwent a second surgery among whom patients had granuloma complicated with chondronecrosis and required totracheotomy due to dyspnea Figure However the diï¬erence between the two groups was not statistically significantp In the PPI treatment group only patients hadtwo unscheduled visits In the control group patients had unscheduled visits among them one patient had visitsdue to dyspnea The diï¬erence in the number of unscheduledvisits was not statistically significant p Table Eï¬ects of PPI Treatment in Patients with LPRD in theExperimental Group Among the patients in the experimental group were diagnosed with LPRD according to preoperative RSI and RFS scores and patients did not have LPRD The numbers of LPRDpatients with granuloma at the 1st 2nd 3rd and 6th monthsafter surgery were and respectively and the numbers of LPRD patients with granuloma at these time pointswere and While the data showed that the percentageof granuloma in the LPRD patients was higher than that inthe LPRD patients only the diï¬erence at the 3rd month aftersurgery was statistically significant p Table Transoral microsurgery TM for earlystage glottic cancerGC can achieve oncological therapeutic eï¬ects similar tothose of radiotherapy TM has the advantages of being minimally invasive and having a high laryngeal preservation rateand a low tracheotomy rate its disadvantage includes postoperative complications such as bleeding infection airwayburns and granuloma formation Therefore currently thetreatment selected for patients with earlystage GC is determined by the disease conditions as well as patient needs[ ] Postoperative traumatic granuloma TG is a common complication during the healing process after TLM surgery Severe TG may cause or be complicated by chondritis orchondronecrosis leading to severe complications includingdyspnea With the increase in the range of cordectomy theincidence of TG is also significantly increased Therefore itis necessary to investigate ways to reduce the incidence andseverity of postoperative TG [ ] Like TLMthecoblationassisted endoscopic cordectomy or TCM used inour study is also based on thermal damage whose working°C much lower than laser™s workingtemperature is °°temperature which is C1000C and as a result its healing process and the mechanism of TG formation are alsosimilar to those of TLM Current literature indicates thatreflux may be an important factor in the occurrence of postoperative TG [ “] Therefore we aimed to investigatewhether antiacid therapy could reduce TG through an analysis of the eï¬ects of PPI treatment in patients who underwenttype IVVI cordectomy which has rarely been reported°C70Our study showed that the incidences of TG after transoral surgery were as high as and in the twogroups which were much higher than the incidences of reported by Nerurkar and Shah and reported by Wang The reason for this discrepancy may be that the patients in our study all underwent typeIV or higher surgeries Enlarged wounds and damage to theperichondrium or cartilage may lead to increased TG andchondronecrosis Nerurkar and Shah reported that the incidence of TG in patients who have undergone type IV TLMwas and the study by Wang showed that the incidences of TG in patients who have undergone type IV andtype V TLM were and respectively [ ]Meanwhile the incidence of chondronecrosis was not rare especially on whom the surgeon had to expose thethyroid cartilage during tumor resection in TLM [] Thesestudies suggest that type IV or higher surgeries lead to a highlikelihood of TG occurrenceOur study showed that PPI treatment did not suppressthe formation of TG at the 1st month after surgery but thepercentage of TG in the PPI treatment group graduallybecame lower than that of the control group and the diï¬erences were statistically significant at the 2nd and 3rd monthsafter surgery At the 6th month granulomas disappeared inboth groups Our findings suggest that although PPI treatment cannot reduce the incidence of TG it can significantlyshorten the duration of granulomas a finding that is similarto the study by Wang [] Additionally we did notobserve any severe cases of TG that were complicated with 0cBioMed Research InternationalabcdefghiFigure a“f Shows a typical case in the control group who was a male patient for years old with glottic cancer T2N0M0 at the left sidefollowed by type V cordectomy a One month after surgery a granuloma was found in the left vocal cord under a fibrolaryngoscope b months after surgery the granuloma enlarged and the right vocal cord become edema obviously c months after surgery d monthsafter surgery e Computerized tomography CT scan taken before surgery f months after surgery chondronecrosis was found in the CTscan where the white arrow points out g“i Shows a typical case in the experimental group who was a male patient for years old withglottic cancer T2N0M0 at the right side followed by type V cordectomy g h and i were taken under a fibrolaryngoscope in month months and months after surgery respectively A granuloma could be found in g but disappeared in h and idyspnea in the PPI treatment group however TG in patients in the control group caused or was complicated bychondritis or chondronecrosis which led to severe dyspneaThere were only unscheduled visits among the PPI treatment group compared to unscheduled visits in the controlgroup Unfortunately the diï¬erences in the two indicators ofTG severity between the two groups were not statistically significant although we believe PPI treatment did alleviate the 0cBioMed Research InternationalTable Percentage and severity of granuloma in the two groups1st month2nd month3rd month6th monthResurgeryNumber of unscheduled visits asmedian and interquartile rangePPI treatment groupControl groupp valueNote aindicates that the diï¬erence is statistically significant0005a0037a Table Percentage of granuloma in patients with or without LPRDin the experimental group1st month 2nd month 3rd month 6th month”LPRD n LPRD n p valueNote aindicates that the diï¬erence is statistically significant0029aseverity of TG In this study postoperative PPI was used for weeks referring to the antiacid duration weeks in amedical routine of vocal process granulation and laryngopharyngeal reflux disease J R Lechien™s report in andChinse experts consensus on diagnosis and treatment of laryngopharyngeal and reflux disease in [] The resultsshowed that PPI could shorten the recovery time and potentially prevent severe complicationsOur study showed that the percentage of TG did not differ significantly between the LPRD and LPRD patientsp However it took longer for granulomas to disappear in LPRD patients than in LPRD patients and the difference in the number of patients with granulomas wasstatistically significant between the two groups at monthsafter surgery p The reason for this phenomenonmay be that during the early stage of recovery after vocalcord injury changes in the extracellular matrix are the mainmanifestation and injury impacts a lot while acid reflux hasa little eï¬ect Acid reflux may impact the repair process in themiddle and late stages [ ]This was a retrospective nonrandomized controlledstudy with a small sample size We only analyzed granulomaformation in the patients and did not assess their oncologicaloutcome We based the diagnosis of LPRD on RSI and RFSscores and did not perform dualprobe 24hour pH monitoring Therefore we were unable to determine whethernonacid reflux had an impact on the formation and development of postoperative granuloma ConclusionThe preventive use of PPI in patients who have undergonetype IVVI cordectomy cannot reduce the incidence of TGwhile it can shorten the TG recovery duration and may alsoreduce the severity of TG Our findings should be confirmedby prospective randomized controlled studies with largersample sizesData AvailabilityAccess to these anonymized data will be made available bythe corresponding author Dr Jian Wang upon reasonablerequestConflicts of InterestThe authors declare that they have no conflicts of interestfinancial or nonfinancial to discloseAuthors™ ContributionsXiaofeng Jin and Yanyan Niu contributed equally to thisstudyAcknowledgmentsThis study was funded by the Nature Science Foundation ofBeijing China Grant No References[] L B Du W M Mao W Q Chen œIncidence and mortality of larynx cancer in China during  ZhonghuaLiu Xing Bing Xue Za Zhi vol no pp “ [] F Bray J Ferlay I Soerjomataram R L Siegel L A Torreand A Jemal œGlobal cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA A Cancer Journal for Clinicians vol no pp “ [] B Gupta N W Johnson and N Kumar œGlobal epidemiology of head and neck cancers a continuing challenge Oncology vol no pp “ [] M F Vaculik C A MacKay S M Taylor J R B Trites R DHart and M H Rigby œSystematic review and metaanalysisof T1 glottic cancer outcomes comparing CO2 transoral lasermicrosurgery and radiotherapy Journal of Otolaryngology Head and Neck Surgery vol no p [] W Steiner œResults of curative laser microsurgery of laryngealcarcinomas American Journal of Otolaryngology vol no pp “ [] M S Strong œLaser excision of carcinoma of the larynxLaryngoscope vol no pp “ [] A S Carney M S Timms C N Marnane S KrishnanG Rees and S Mirza œRadiofrequency coblation for the resection of head and neck malignancies Otolaryngology and Headand Neck Surgery vol no pp “ [] M Lee M A Buchanan F Riï¬at and C E Palme œComplications after CO2 laser surgery for early glottic cancer an 0cBioMed Research Internationalinstitutional experience Head Neck vol no S1 pp E987“E990 [] B Liu L Cheng H Ming and C Zhong œTreatment of theearlystage glottic cancer using lowtemperature radiofrequency coblation Journal of Cancer Research and Therapeutics vol no pp “ [] Y Zhang B Wang G Sun G Zhang L Lu and G LiangœCarbon dioxide laser microsurgery versus lowtemperatureplasma radiofrequency ablation for T1a glottic cancer asingleblind randomized clinical trial BioMed Research International vol Article ID pages [] M Remacle H E Eckel A Antonelli œEndoscopic cordectomy A proposal for a classification by the Working Committee European Laryngological Society European Archivesof OtoRhinoLaryngology vol no pp “ [] L Wang S Sun S Wang D Liang and W Ji œClinical observation of traumatic granuloma after CO‚‚ laser cordectomy andlaryngopharyngeal reflux Zhonghua Er Bi Yan Hou Tou JingWai Ke Za Zhi vol no pp “ [] M Canis F Ihler A Martin C Matthias and W SteinerœTransoral laser microsurgery for T1a glottic cancer reviewof cases Head Neck vol no pp “ [] A Galli L Giordano D Sarandria D di Santo and M BussiœOncological and complication assessment of CO2 laserassisted endoscopic surgery for T1T2 glottic tumours clinicalexperience Acta Otorhinolaryngologica Italica vol no pp “ [] N K Nerurkar and R Shah œFactors responsible for the development of carbon granuloma post transoral laser cordectomy Lasers in Medical Science vol no pp “ [] P C Belafsky G N Postma and J A Koufman œThe validityand reliability of the reflux finding score RFS Laryngoscopevol no pp “ [] P C Belafsky G N Postma and J A Koufman œValidity andreliability of the reflux symptom index RSI Journal of Voicevol no pp “ [] M Remacle C van Haverbeke H Eckel œProposal forrevision of the European Laryngological Society classificationof endoscopic cordectomies European Archives of OtoRhinoLaryngology vol no pp “ [] J Yoo C Lacchetti J A Hammond R W Gilbert and Headand Neck Cancer Disease Site Group œRole of endolaryngealsurgery with or without laser versus radiotherapy in themanagement of early T1 glottic cancer a systematic reviewHead Neck vol no pp “ [] C M Chiesa Estomba F A Reinoso A O Velasquez J LFernandez J L Conde and C S Hidalgo œComplications inCO2 laser transoral microsurgery for larynx carcinomas IntArch Otorhinolaryngol vol no pp “ [] J R Lechien F Mouawad M R Barillari œTreatment oflaryngopharyngeal reflux disease a systematic review WorldJournal of Clinical Cases vol no pp “ [] M K Wani and G E Woodson œLaryngeal contact granuloma Laryngoscope vol no pp “ [] C Ling M Yamashita J Zhang D M Bless and N V Welham œReactive response of fibrocytes to vocal fold mucosalinjury in rat Wound Repair and Regeneration vol no pp “ 0c'
2
"To investigate the efficacy and safety of percutaneous microwave ablation. Methods: Twenty-six rabbits with lung VX2 tumor were randomly divided into experimental and control group. In the experimental group microwave ablation guided by ultrasound or CT was performed based on location of the tumor. Enhanced CT scan was carried out immediately before and after the ablation for all animals. Two animals from each group were sacrificed immediately or 1 week after the ablation respectively and the others were followed for the rest of their lives. Results: CT scan revealed that the tumor was greatly reduced or ablated after ablation. Pathological examination immediately after ablation also confirmed the tumor reduction or ablation. The survival time of the animals in the experimental group was significantly longer than that in the control group. Conclusions: Microwave ablation is a safe and effective method for treating lung cancer in rabbits showing potential clinical applicability. Microwave ablation VX2 tumor lung cancer 9421547 4136 Hum Pathol Hum. Pathol. Human pathology 0046-8177 1532-8392 24444464 3965626 10.1016/j.humpath.2013.10.016 NIHMS537247 A PIK3CA mutation detected in plasma from a patient with synchronous primary breast and lung cancers Jelovac Danijela MD 1 * Beaver Julia A. MD 1 * Balukrishna Sasidharan MD 2 Wong Hong Yuen BS 1 Toro Patricia Valda BS 1 Cimino-Mathews Ashley MD 1 Argani Pedram MD 1 Stearns Vered MD 1 Jacobs Lisa MD 1 VanDenBerg Dustin BS 1 Kessler Jill BS 1 Jeter Stacie BS 1 Park Ben H. MD PhD 1 Wolff Antonio C. MD 1 1The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 1650 Orleans Street Baltimore MD 21287 2Christian Medical College Vellore Tamil Nadu India 632004 * These authors contributed equally to this work 18 12 2013 31 10 2013 4 2014 01 4 2015 45 4 880 883 2013 Elsevier Inc. All rights reserved. 2013 Digital PCR is a new technology that enables detection and quantification of cancer DNA molecules from peripheral blood. Using this technique we identified mutant PIK3CA DNA in circulating plasma tumor DNA (ptDNA) from a patient with concurrent early stage breast cancer and non-small cell lung cancer. The patient underwent successful resection of both her breast and lung cancers and using standard Sanger sequencing the breast cancer was shown to harbor the identical PIK3CA mutation identified in peripheral blood. This case report highlights potential applications and concerns that can arise with the use of ptDNA in clinical oncology practice. plasma tumor DNA breast cancer lung cancer PIK3CA digital PCR Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24983368 4102953 bjc2014353 10.1038/bjc.2014.353 Genetics and Genomics Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing Worldwide external quality assessment for EGFR gene mutation testing Patton S 1 * Normanno N 2 Blackhall F 3 Murray S 4 Kerr K M 5 Dietel M 6 Filipits M 7 Benlloch S 8 Popat S 9 Stahel R 10 Thunnissen E 11 1EMQN Manchester Centre for Genomic Medicine St Mary's Hospital Manchester M13 9WL UK 2Cell Biology and Biotherapy Unit Istituto Nazionale per lo Studio e la Cura dei Tumori ˜Fondazione Giovanni Pascale'”IRCCS 80131 Naples Italy 3Christie Hospital Manchester M20 4BX UK 4Biomarker Solutions Ltd London EC1V 2NX UK 5Department of Pathology Aberdeen Royal Infirmary Aberdeen AB25 2ZN UK 6Charit Humboldt-Universitt zu Berlin Berlin 10117 Germany 7Medical University of Vienna 1010 Vienna Austria 8Pangaea Biotech USP Dexeus University Institute Barcelona 08028 Spain 9Royal Marsden Hospital London SW3 6JJ UK 10University Hospital Z¼rich CH-8091 Z¼rich Switzerland 11Department of Pathology VU University Medical Center Amsterdam 1081 HZ The Netherlands *E-mail: simon.pattoncmft.nhs.uk 15 07 2014 01 07 2014 15 7 2014 111 2 413 420 22 01 2014 19 05 2014 20 05 2014 Copyright 2014 Cancer Research UK 2014 Cancer Research UK This work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-sa/3.0/ Background: The external quality assurance (EQA) process aims at establishing laboratory performance levels. Leading European groups in the fields of EQA Pathology and Medical and Thoracic Oncology collaborated in a pilot EQA scheme for somatic epidermal growth factor receptor (EGFR) gene mutational analysis in non-small-cell lung cancer (NSCLC). Methods: EQA samples generated from cell lines mimicking clinical samples were provided to participating laboratories each with a mock clinical case. Participating laboratories performed the analysis using their usual method(s). Anonymous results were assessed and made available to all participants. Two subsequent EQA rounds followed the pilot scheme. Results: One hundred and seventeen labs from 30 countries registered and 91 returned results. Sanger sequencing and a commercial kit were the main methodologies used. The standard of genotyping was suboptimal with a significant number of genotyping errors made. Only 72 out of 91 (72%) participants passed the EQA. False-negative and -positive results were the main sources of error. The quality of reports submitted was acceptable; most were clear concise and easy to read. However some participants reported the genotyping result in the absence of any interpretation and many obscured the interpretation required for clinical care. Conclusions: Even in clinical laboratories the technical performance of genotyping in EGFR mutation testing for NSCLC can be improved evident from a high level of diagnostic errors. Robust EQA can contribute to global optimisation of EGFR testing for NSCLC patients. non-small-cell lung carcinoma EGFR gene mutations quality assessment Assessment of epidermal growth factor receptor (EGFR) mutations has become mandatory to choose the most active first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC). Indeed randomized phase III clinical trials have demonstrated that first-line administration of an EGFR TKI results in a prolonged progression-free survival as compared with chemotherapy in patients carrying EGFR mutations (Mok et al 2009; Maemondo et al 2010; Mitsudomi et al 2010; Fukuoka et al 2011; Zhou et al 2011; Rosell et al 2012). These studies have also confirmed that EGFR mutations are a reliable marker that predicts sensitivity to EGFR TKIs (Mok et al 2009). Activating mutations occur in exons 18 through 21 of the TK domain of the EGFR gene and either point mutations or in-frame small deletions or insertions (Sharma et al 2007; De Luca and Normanno 2010). Although more than 250 mutations of the EGFR gene have been described to date two mutations a single point mutation in exon 21 the L858R and a series of small in-frame deletions in exon 19 account for ?90% of all EGFR mutations (Sharma et al 2007; Linardou et al 2008). EGFR mutations are strongly associated with defined clinical and pathological features: they are far more frequent in female patients as compared with male; in adenocarcinoma as compared with other histological types; in non-smokers as compared with current smokers or former smokers; and in East-Asian NSCLC patients as compared with Non-East-Asian patients (Normanno et al 2006). External quality assessment (EQA) is a system of objectively checking laboratory results by an independent external agency (van Krieken et al 2013). The main objective of an EQA programme is to establish inter-laboratory comparability. In this respect the EQA process can identify latent systematic errors in methodology that may not be revealed by a laboratory's own internal QA processes. Representatives from ETOP ESMO ESP EMQN and other leading European groups met in July 2010 to discuss a pan-European approach to EQA for EGFR mutation testing in NSCLC. In this paper we present the results of this pilot EQA scheme for EGFR testing that was completed in 2013. Materials and Methods anisation of the scheme A meeting was anised in July 2010 by ETOP and EMQN to bring together a group of professionals representing EMQN ESP ETOP ESMO and other leading European groups involved in NSCLC testing (see Supplementary Information). From this group a steering group of five individuals was formed who planned designed and assessed the results of the pilot EQA scheme. The scheme was coordinated and administered by the EMQN and three rounds were anised within a period of 18 months. The workflow of the scheme process is shown in Figure 1."
1
" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to 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 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the œconsensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from ˆ’ to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as œphagosomeœautoimmune thyroid disease œantigen processing andpresentation œb cell receptor signaling pathway œintestinal immune network for iga production œinflammatorybowel disease œprimary immunodeficiency œth1 andth2 cell differentiation œth17 cell differentiation œnatural killer cell mediated cytotoxicity and œnfˆ’kappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors™ contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didn™t involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am “biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw “simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol “huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem “liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg “xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett “zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med “ binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med“ yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol “ deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med “ pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a
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"Long non‘coding RNA FOXD2‘AS1 regulates the tumorigenesis and progression of breast cancer via the S100 calcium binding protein A1Hippo signaling pathwayPEI HUANG1 and JINHUI XUE21Department of Pathology The First Affiliated Hospital of Zhengzhou University Zhengzhou Henan 2Department of Pathology Zhengzhou Central Hospital Affiliated to Zhengzhou University Zhengzhou Henan PR ChinaReceived November Accepted April 103892ijmm20204699Abstract Breast cancer is one of the most prevalent cancer types and is accompanied by a high incidence and mortality rate severely threatening women's health globally Long noncoding RNA forkhead box d2 adjacent apposite strand RNA lncRNA FOXD2‘AS1 has been identified to function as an oncogene in human cancers however it has rarely been investigated in breast cancer The aim of the present study was to investigate the role of FOXd2AS1 in breast cancer and to clarify the underlying mechanisms The expression of FOXD2‘AS1 in breast cancer cell lines was first quantified by reverse transcription‘quantitative PCR and the biological function of FOXD2‘AS1 was then determined Cellular proliferative ability was determined by Cell Counting kit‘ assay and wound healing and Transwell assays were conducted to assess the cell migratory and invasive ability corresponding protein expression levels were determined by western blot analysis In addition experimental animal models were established by the subcutaneous injection of MDA‘MB‘ cells into the right axillary lymph nodes of BALBc nude mice and the effects of FOXD2‘AS1 on tumor growth were observed The results indicated that FOXD2‘AS1 expression was upregulated in breast cancer cell lines and that FOXD2‘AS1 downregulation significantly inhibited the proliferation migration and invasiveness of MCF‘ and MDA‘MB‘ cells S100 calcium binding protein A1 S100A1 was also upregulated in breast cancer cell lines and was positively regulated by FOXD2‘AS1 Furthermore the inhibition of S100A1 and the overexpression of the serinethreonine‘protein kinase large tumor suppressor homolog LATS1 inhibited the FOXD2‘AS1‘induced Correspondence to Dr Pei Huang Department of Pathology The First Affiliated Hospital of Zhengzhou University East Jianshe Road Zhengzhou Henan PR ChinaE‘mail huangpei193163comKey words FOXD2‘AS1 S100 calcium binding protein A1 breast cancer large tumor suppressor homolog Hippocellular proliferation migration and invasiveness in breast cancer Experimental mouse models revealed that FOXD2‘AS1 downregulation significantly inhibited tumor growth and that the levels of phosphorylated p‘YAP and p‘LATS1 were upregulated by FOXD2‘AS1 knockdown indicating that the inhibition of FOXd2AS1 activated Hippoyesassociated protein signaling On the whole the findings of the present study suggest that the FOXD2‘AS1S100A1Hippo axis is involved in the tumorigenesis and progression of breast cancer In the future these may contribution to the identification of more effective breast cancer treatmentsIntroductionAs one of the most prevalent malignancies breast cancer is a primary cause of mortality among gynecological cancer cases and with increasing morbidity and mortality rates it poses a considerable threat to women's health worldwide In statistics from the American cancer Society estimated newly diagnosed cases and deaths from breast cancer in the United States The leading causes of the high death rate are distal metastasis and resistance to the existing treatments despite improvements in early diagnosis and systemic treatment the incidence of breast cancer and metastasisrelated mortality is steadily increasing Therefore there is an urgent need to elucidate the mechanisms responsible for the disordered cellular metastasis and to enhance our understanding of the tumorigenesis and development processes hence facilitating the identification of more efficient breast cancer treatmentsLong noncoding RNAs lncRNAs are a group of RNAs nucleotides in length which lack protein‘coding capacity Numerous studies have revealed that lncRNAs have versatile biological functions in pathological and physiological processes including tumorigenesis ‘ lncRNAs are considered to regulate the development of various types of cancer including breast cancer For instance LINC01089 is downregulated in breast cancer tissues and cell lines and LINC01089 overexpression increases tumor cell proliferation migration and invasiveness As an oncogene that regulates breast cancer cell proliferation and apoptosis hepatocellular 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERcarcinoma upregulated EZH2‘associated lncRNA is closely associated with the clinical progression of breast cancer These results indicate the indispensability of research on lncRNAs and breast cancerlncRNA forkhead box d2 adjacent apposite strand RNA FOXD2‘AS1 is a novel non‘coding RNA identified to be an oncogene in human cancers FOXD2‘AS1 has been shown to be upregulated in various types of cancer including glioma osteosarcoma and papillary thyroid cancer as well as breast cancer ‘ A previous study indicated that FOXD2‘AS1 participates in regulating the development of breast cancer via the miR‘‘5pPFN2 axis and that it may be a potential biomarker for the diagnosis and prognosis of breast cancer However to the best of our knowledge there are no additional data regarding the investigation of FOXd2AS1 in breast cancer and its effects and the underlying mechanisms on the regulation of breast cancer cell invasion and metastasis Thus the aim of the present study was to determine the role and potential mechanisms of action of FOXd2AS1 in breast cancer and to provide further support for its use in clinical diagnosis and treatmentMaterials and methodsDatasets The present study evaluated the expression level of FOXD2‘AS1 in breast cancer samples using The Cancer Genome Atlas TCGA dataset which was downloaded from the TCGA data portal tcga‘data‘nci‘nih‘govezxjtlueducn The TCGA data subset for breast cancer included normal samples and tumor samples The Mann‘Whitney test was used to determine statistically significant differences between normal and tumor samples P005 was considered to indicate a statistically significant differenceCell culture A human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ were purchased from the American Type Culture Collection ATCC The cells were incubated in RPMI‘ medium supplemented with fetal bovine serum FBS in a humidified incubator at ˚C CO2 Transfection To overexpress FOXd2AS1 an overexpression vector pcdNA FOXd2AS1 and its corresponding negative control vector pcDNA‘NC were synthesized by Shanghai GenePharma Co Ltd Short hairpin shRNAs targeting FOXD2‘AS1 nM shRNA‘FOXD2‘AS1‘ and shRNAFOXd2AS12 and a negative scramble control shRNA shRNA also purchased from Shanghai GenePharma Co Ltd were used to knock down FOXD2‘AS1 expression In addition pcdNALATS1 shRNAS100A11 and shRNAS100A12 were obtained from Shanghai GenePharma Co Ltd to overexpress LATS1 or to knock down S100A1 respectively The shRNA sequences were as follows shRNA‘FOXD2‘AS1‘ targets GGA CTC CAC TCT TCG CTT A shRNA‘FOXD2‘AS1‘ targets GCT TCC AGG TAT GTG GGA A shRNA‘S100A1‘ targets GAT CCG GAG ACC CTC ATC AAC GTG TTC TTC CTG TCA GAA ACA CGT TGA TGA GGG TCT CCT TTT TG shRNA‘S100A1‘ targets GAT CCG TGG ACT TCC AGG AGT ATG TGC TTC CTG TCA GAC ACA TAC TCC TGG AAG TCC ACT TTT TG Cells were transfected with pcDNA FOXD2‘AS1 nM pcdNALATS1 nM pcdNANc nM shRNAFOXd2AS11 ngµl shRNAFOXd2AS12 ngµl shRNAS100A11 ngµl shRNAS100A12 ngµl shRNA ngµl or co transfected with pcdNA FOXd2AS1 and pcdNALATS1 or cotransfected with pcDNA FOXD2‘AS1 and shRNA‘S100A1 using Lipofectamine® transfection reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instructions Lipofectamine reagent was first mixed with vectors to form a reagent‘vector complex followed by incubation with cells at ˚C for h The transfection efficacy was assessed by reverse transcription‘quantitative PCR RT‘qPCR after h of transfectionRNA extraction and RT‘qPCR Total RNA was extracted from all cell lines using TRIzol® reagent Takara Bio Inc according to the manufacturer's instructions Total RNA was then reverse transcribed into cDNA using the PrimeScript„¢ RT Master kit and the mRNA expression levels were quantified using the SYBR Premix Ex Taq„¢ kit Both Takara Bio Inc with a Fast Real‘Time PCR System Applied Biosystems Thermo Fisher Scientific Inc The sequences of specific primers used for RT‘qPCR were as follows FOXDA‘AS1 forward '‘TGG ACC TAG CTG CAG CTC CA‘' and reverse '‘AGT TGA AGG TGC ACA CAC TG‘' S100A1 forward '‘GAG TAT GTG GTG CTT GTG GC‘' and reverse '‘CTT GGA CCG CTA CTC TTG CG‘' large tumor suppressor homolog LATS1 forward '‘ACC GCT TCA AAT GTG ACT GTG ATG CCA C CT‘' and reverse '‘CTT CCT TGG GCA AGC TTG GCT GAT CCT CT‘' and GAPDH forward '‘GCG AGA TCG CAC TCA TCA TCT ‘' and reverse '‘TCA GTG GTG GAC CTG ACC ‘' The data were displayed as ‘ΔΔCq values with GAPDH as the constitutive marker The PCR conditions were as follows ˚C for min cycles of ˚C for sec and ˚C for sec followed by ˚C for minCell Counting Kit‘ CCK‘ assay cell proliferation was determined using the Cell Counting Kit‘ assay CCK‘ Dojindo Molecular Technologies Inc Briefly cells were seeded into a ‘well plate and incubated for h at ˚C Following culture for the indicated periods of time and h µl of the CCK‘ reagent were added to each well and the plate was incubated at ˚C for a further h The optical density values at nm were then measured using a microplate spectrophotometer Thermo Fisher Scientific IncWestern blot analysis The total protein was extracted from the cells using RIPA lysis buffer Beyotime Institute of Biotechnology and quantified using a BCA protein assay kit Thermo Fisher Scientific Inc The same amount of each protein sample µg was subjected to SDS‘PAGE the proteins were then transferred onto PVDF membranes EMd Millipore and was blocked in nonfat milk for h at room temperature The membranes were then incubated with primary antibodies against cyclinE1 cat no ab33911 Abcam cyclin‘dependent kinase‘ CDK2 cat no ab32147 Abcam p21 cat no ab109520 Abcam matrix metalloproteinases MMP2 cat no ab92536 Abcam MMP9 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE was added to the lower 24well chamber After h cells on the upper surface were removed and cells attached to the lower surface were stained with crystal violet Beijing Solarbio Science Technology Co Ltd at room temperature for min The cells were viewed under a light microscope magnification x100 CKX41 Olympus Corporation and the invasive ability of the cells was determined using ImageJ software version by counting the number of cells attached to the lower surfaceCell cycle analysis The cell cycle distribution was determined by flow cytometry After being subjected to the indicated treatments the cells were collected and fixed in ethanol at ‘ËšC overnight The cells were then washed twice with PBS and incubated in the dark with RNase A and PI staining solution Roche Diagnostics at ˚C for min Finally the cell samples were analyzed using a FACSCalibur flow cytometer and CellQuest software version both from BD BiosciencesIn vivo experiments The present study was approved by the First Affiliated Hospital of Zhengzhou University and the animal experiments were performed according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals A total of 5weekold male BALBc nude mice were purchased from the Experimental Animal Center of Shanghai Institute for Biological Sciences and housed in a standard environment ˚C humidity h lightdark cycle with free access to food and water Each mouse was subcutaneously injected at the right axillary lymph nodes with 10x107 MdAMB468 cells which were stably transfected with either the shRNA negative control shRNA or shRNA‘FOXD2‘AS1 The weights and tumor volumes tumor volume x length x width2 of the mice were monitored every days until the mice were sacrificed At days after the injection all the mice were sacrificed by cervical dislocation that caused a sharp section of the spinal cord followed by an instantaneous cardiac arrest After the cessation of the heartbeat and respiratory arrest of the mice was confirmed the tumors were excised photographed and stored for the further investigationStatistical analysis data are presented as the means ± standard deviation SD from ‰¥ independent experiments and each experiment was conducted in triplicate The data were analyzed using SPSS statistical software version SPSS Inc and the differences among groups were analyzed using one‘way analysis of variance followed by Tukey's post hoc test P005 was considered to indicate a statistically significant differenceResultsFOXD2‘AS1 expression is upregulated in breast cancer cells The TCGA database cancergovtcga was used to identify the association between FOXD2‘AS1 and breast cancer by evaluating the expression profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues The results of TCGA analysis revealed a significantly higher FOXd2AS1 expression in breast cancer tissues than in normal tissues Fig 1A Human Figure FOXD2‘AS1 is upregulated in breast cancer cells A The Cancer Genome Atlas TCGA database cancergovtcga was used to identify the association of FOXD2‘AS1 with breast cancer by collecting the profiles of FOXD2‘AS1 in breast cancer tissues and normal tissues P0001 vs normal samples B mRNA level of FOXD2‘AS1 in human normal breast epithelial cell line MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P001 and P0001 vs MCF‘10A cells cat no ab38898 Abcam S100 calcium binding protein A1 S100A1 cat no Cell Signaling Technology Inc phosphorylated p‘yes‘associated protein YAP cat no Cell Signaling Technology Inc YAP cat no Cell Signaling Technology Inc serinethreonine‘protein kinase LATS1 cat no Cell Signaling Technology Inc p‘LATS1 cat no Cell Signaling Technology Inc mammalian STE20‘like protein kinase MST1 cat no Cell Signaling Technology Inc MST2 cat no Cell Signaling Technology Inc and GAPDH cat no ab8245 Abcam at ˚C overnight The membranes were washed with Tris‘buffered saline with Tween TBST and incubated with horseradish peroxidase HRP‘conjugated goat anti‘mouse IgG secondary antibody cat no sc‘ Santa Cruz Biotechnology Inc at room temperature for h The protein bands were visualized using an enhanced chemiluminescence kit Amersham Pharmacia Biotech and quantified using ImageJ software version National Institutes of HealthWound healing assay The cellular migration rate was determined using a wound healing assay The cells were seeded into a ‘well plate and cultured to confluence A wound was produced in each monolayer using a ‘µl pipette tip and the plate was washed times with PBS to remove detached cells The cells were then cultured in the fresh medium without FBS Following incubation for h the wound‘healing ability was assessed under a light microscope magnification x100 CKX41 Olympus Corporation and the widths of the wounds were measured at and hTranswell assay The cell invasive rate was determined with a Transwell assay cells 4x104well in serum‘free medium were placed in the upper chamber of each insert [which had been precoated with µl of Matrigel Bd biosciences at ˚C for h] and complete medium containing FBS 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer A and B Following transfection with shRNA‘FOXD2‘AS the mRNA level of FOXD2‘AS1 was measured by RT‘qPCR in MCF‘ and MDA‘MB‘ cells C and D CCK‘ assay was performed to determine cell proliferation following transfection E‘G Cell cycle distribution was determined and analyzed by FACS H and I The protein expression of cyclin E1 CDK2 and p21 was determined by western blot analysisnormal breast epithelial cell line MCF‘10A and human breast cancer cell lines McF7 MdAMB468 MdAMB453 and BT549 were also obtained to detect the mRNA levels of FOXD2‘AS1 The results revealed that FOXD2‘AS1 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Continued Knockdown of FOXD2‘AS1 suppresses cell proliferation migration and invasion in breast cancer J Wound healing assay was performed to detect the migration of both MCF‘ and MDA‘MB‘ cells K and L Relative migration rate of MCF‘ and MDA‘MB‘ cells was quantified respectively M Transwell assay was performed to detect the invasion of both McF7 and MdAMB468 cells N and O Relative cell invasive rate of McF7 and MDA‘MB‘ cells was quantified respectively P MMP‘ and MMP‘ protein expression in MCF‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified Q MMP‘ and MMP‘ protein expression in MDA‘MB‘ transfected with shRNA‘FOXD2‘AS1 was detected and quantified P005 P001 and P0001 vs shRNA‘NC CDK2 cyclin‘dependent kinase‘ MMP matrix metalloproteinase expression was markedly upregulated in all breast cancer cells particularly in the MCF‘ ER‘positive breast cancer cell line and MdAMB468 cells triplenegative breast cancer cell line Fig 1B which were used for further experiments even 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 regulates the HippoYAP signaling pathway A The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MCF‘ cells B The protein expression of HippoYAP signaling pathway‘related genes p‘YAP YAP p‘LATS1 LATS1 MST1 and MST2 was determined by western blot analysis in MDA‘MB‘ cells P005 P001 and P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog MST mammalian STE20‘like protein kinase YAP yes‘associated protein though there may be some variability in the results between the cell lines These findings indicate that FOXD2‘AS1 is upregulated in breast cancerFOXD2‘AS1 knockdown suppresses breast cancer cell proliferation migration and invasiveness To further elucidate the role of FOXd2AS1 in breast cancer FOXd2AS1 was knocked down in both the McF7 and MdAMB468 cells Due to a higher transfection efficacy shRNA‘FOXD2‘AS1‘ referred to as shRNA‘FOXD2‘AS1 was subsequently used for breast cancer cell experimentation Fig 2A and B The results of CCK‘ assay indicated that FOXD2‘AS1 knockdown significantly inhibited the proliferative ability of the MCF‘ and MdAMB468 cells Fig 2c and d The cell cycle was then analyzed by flow cytometry which revealed that FOXd2AS1 knockdown increased the percentage of cells in the G1 phase whereas it decreased that in the S phase for both the MCF‘ and MDA‘MB‘ cells Fig 2E‘G Furthermore FOXd2AS1 knockdown decreased the protein expression levels of cyclin E1 and CDK2 and increased the expression of 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure S100A1 is upregulated in breast cancer cells A The mRNA level of S100A1 in MCF‘10A and human breast cancer cell lines MCF‘ MDA‘MB‘ MDA‘MB‘ and BT‘ was determined by RT‘qPCR P005 P001 and P0001 vs MCF‘10A cells B and C In shRNA‘FOXD2‘AS1‘transfected McF7 and MdAMB468 cells the protein expression of Sl00A1 was detected by western blot analysis P005 and P0001 vs shRNA‘NC D and E Following transfection with pcDNA‘FOXD2‘AS1 the mRNA level of FOXD2‘AS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR P001 and P0001 vs pcDNA‘NC MCF‘ and MDA‘MB‘ cells were transfected with shRNA‘S100A1 and the protein expression and mRNA level of S100A1 in F and G MCF‘ and H and I MDA‘MB‘ cells were determined by western blot analysis and RT‘qPCR respectively P001 and P0001 vs shRNA‘NC S100A1 S100 calcium binding protein A1 p21 Fig 2H and I These findings indicate that FOXD2‘AS1 knockdown suppresses cellular proliferation by regulating the cell cycle specifically by preventing G1 to S phase progression Moreover FOXD2‘AS1 knockdown significantly decreased the migration rate Fig 2JL and the invasiveness Fig 2MO of the McF7 and MdAMB468 cells The 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure S100A1 mediates FOXD2‘AS1‘induced cell proliferation migration and invasion in breast cancer Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with shRNA‘S100A1 A and B CCK‘ assay was performed to determine the proliferation of the differently treated cells C Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay D Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay E Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P001 and P0001 vs FOXD2‘AS1 shRNA‘NC S100A1 S100 calcium binding protein A1 expression levels of MMP‘ and ‘ which are critical to the migration invasion and metastasis of breast cancer cells were both downregulated following FOXD2‘AS1 knockdown Fig 2P and Q indicating that FOXD2‘AS1 may enhance cellular migration and invasiveness by regulating MMP‘ and ‘FOXD2‘AS1 knockdown regulates the HippoYAP signaling pathway The HippoYAP signaling pathway is reportedly involved in the progression of breast cancer In the present study the levels of specific proteins involved in the YAPHippo signaling pathway were assessed in the MCF‘ and MdAMB468 cells following FOXd2AS1 knockdown Western blot analysis revealed that the levels of p‘YAP and p‘LATS1 were significantly upregulated while those of MST1 and were significantly downregulated by FOXD2‘AS1 knockdown Fig 3A and B Thus the results confirmed that FOXD2‘AS1 regulates the HippoYAP signaling pathway in breast cancer cellsS100A1 mediates FOXD2‘AS1‘induced breast cancer cell proliferation migration and invasiveness S100A1 is a 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE Figure Overexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasion A and B mRNA level of LATS1 in MCF‘ and MDA‘MB‘ cells was detected by RT‘qPCR in LATS1‘overexpressing cells P0001 vs overexpression‘NC oe‘NC Following transfection with pcDNA‘FOXD2‘AS1 cells were transfected with pcDNA‘LATS1 to overexpress LATS1 C and D CCK‘ assay was performed to determine the proliferation of the differently treated cells E Migratory ability of MCF‘ cells in the different treatment groups was determined by wound healing assay F Invasive ability of MCF‘ cells in the different treatment groups was determined by Transwell assay G Migratory ability of MDA‘MB‘ cells in the different treatment groups was determined by wound healing assay H Invasive ability of MDA‘MB‘ cells in the different treatment groups was determined by Transwell assay P0001 vs pcDNA‘NC P0001 vs FOXD2‘AS1 shRNA‘NC LATS1 large tumor suppressor homolog calcium‘binding protein of the S100 protein family which is not only upregulated in but is also involved in the progression of ovarian cancer In the present study the expression of S100A1 was evaluated in the breast cancer cell lines indicating that S100A1 was significantly upregulated in breast cancer cells particularly in the MCF‘ and MDA‘MB‘ cells compared with the McF10A cells Fig 4A In McF7 and MdAMB468 cells transfected with shRNAFOXd2AS1 it was found that the protein expression level of S100A1 was downregulated Fig 4B and C To further investigate the role of S100A1 in FOXD2‘AS1‘mediated cellular proliferation migration and invasiveness breast cancer cells were transfected with an expression vector pcdNAFOXd2AS1 Fig 4d and E and shRNAS100A1 to inhibit S100A1 protein and mRNA expression Fig 4FI As shown in Fig 5A and B the overexpression of FOXd2AS1 significantly promoted the proliferation of McF7 and MdAMB468 cells which was subsequently reversed by the downregulation of S100A1 0cHUANG and XUE DOWNREGULATION OF FOXD2‘AS1 INHIBITS THE TUMORIGENESIS OF BREAST CANCERFigure Knockdown of FOXD2‘AS1 suppresses tumor progression of breast cancer in vivo A Each mouse was injected with 10x107 MdAMB468 cells which were stably transfected with either shRNA negative control or shRNA‘FOXD2‘AS1 subcutaneously at the right axillary lymph node Following sacrifice the tumors were excised and photographed B and C During the experiment the mouse weight and tumor volume tumor volume x length x width2 was monitored every days until the mice were sacrificed D Protein expression levels of p‘YAP YAP p‘LATS1 and LATS1 in the extracted tumors were determined by western blot analysis and were then quantified P0001 vs shRNA‘NC LATS1 large tumor suppressor homolog YAP yes‘associated protein Wound healing and Transwell assays demonstrated that FOXD2‘AS1 overexpression significantly increased MCF‘ cell migration and invasiveness respectively which were also reversed by the downregulation of S100A1 Fig 5C and D A similar result was observed in the MDA‘MB‘ cells Fig 5E and F These results suggest that S100A1 regulates the FOXd2AS1mediated proliferation migration and invasiveness of breast cancer cellsOverexpression of LATS1 inhibits FOXD2‘AS‘induced cell proliferation migration and invasiveness Since the HippoYAP signaling pathway is involved in the FOXd2AS1mediated characteristics of breast cancer cells HippoYAP signaling was further investigated for its regulatory role in breast cancer cell proliferation migration and invasiveness For this purpose LATS1 was overexpressed in the McF7 and MdAMB468 cells Fig 6A and B and the results of CCK‘ assay revealed that LATS1 overexpression significantly inhibited FOXD2‘AS1‘induced cellular proliferation Fig 6C and D Furthermore the results of wound healing and Transwell assays revealed that LATS1 overexpression significantly inhibited the FOXD2‘AS1‘induced migration and invasiveness of both the McF7 Fig 6E and F and MDA‘MB‘ cells Fig 6G and HFOXD2‘AS1 knockdown suppresses breast cancer tumor progression in vivo From the aforementioned results the role of FOXd2AS1 in both the McF7 and MdAMB468 cells was confirmed To explore the role of FOXD2‘AS1 in breast cancer in vivo mice were injected with MdAMB468 cells which were stably transfected with either an shRNA negative control or shRNA‘FOXD2‘AS1 Following sacrifice 0cINTERNATIONAL JOURNAL OF MOLEcULAR MEdIcINE the tumors were excised and photographed Fig 7A Tumors in the shRNA‘FOXD2‘AS1 group were the smallest in size directly reflecting the suppressive effect of FOXD2‘AS1 knockdown on tumor growth During the experiment body weights and tumor volumes were recorded every days Body weight increased at a slower rate in the shRNAFOXd2AS1 group and the tumor volumes of this group also increased at a slower rate than those in the other groups Fig 7B and C Additionally western blot analysis of the extracted tumor tissues indicated a significant increase in p‘YAP and p‘LATS1 expression in the shRNA‘FOXD2‘AS1 group Fig 7D which was consistent with the in vitro results These findings thus suggest that FOXD2‘AS1 knockdown suppresses the progression of breast cancer and regulates the HippoYAP signaling pathway in vivoDiscussionBreast cancer is one of the most common types of human tumors particularly among females The high rates of metastasis and recurrence typically result in the deterioration and death of patients with breast cancer Increasing evidence suggests that lncRNAs function as oncogenic or antitumor genes in various tumor types cells types and the microenvironment and that they may be used as effective and specific biomarkers for clinical diagnosis and prognosis Due to its oncogenic properties lncRNA FOXd2AS1 has been investigated in several malignant tumors In the present study FOXD2‘AS1 expression was found to be upregulated in breast cancer cell lines thus it was knocked down in MCF‘ and MdAMB468 cell to investigate its role in breast cancer FOXd2AS1 knockdown inhibited the proliferation migration and invasiveness of McF7 and MdAMB468 cells and inhibited tumor growth in vivo Notably S100A1 expression was also found to be upregulated in breast cancer cells and further investigation revealed that S100A1 was inhibited following FOXd2AS1 knockdown indicating that the expression of FOXd2AS1 and S100A1 was positively associated in breast cancer cells Subsequent experiments revealed that the overexpression of FOXD2‘AS1 significantly accelerated tumorigenesis by promoting cellular proliferation migration and invasiveness However the effects of FOXd2AS1 were reversed by the downregulation of S100A1 These results suggest that both FOXD2‘AS1 and S100A1 knockdown exert antitumor effects on the progression of breast cancer and that FOXD2‘AS1 may exert its oncogenic functions by regulating S100A1S100A1 is a calcium‘binding protein belonging to the S100 protein family which exhibit a range of biological properties surrounding cellular proliferation metastasis immune evasion and angiogenesis and are also involved in tumorigenesis For example S100A4 enhances p53dependent apoptosis and facilitates more aggressive tumor progression S100A6 has been reported to be upregulated in human osteosarcoma colorectal carcinoma and hepatocellular carcinoma which was mostly associated with its suppressive properties towards cancer cell migration and tumor metastasis Therefore S100 proteins play an important role in the development and progression of tumors highlighting the necessity to further understand their roles and potential underlying mechanisms In the present study S100A1 expression was found to be upregulated in breast cancer cell lines FOXd2AS1 overexpression was shown to accelerate breast cancer progression by promoting cellular proliferation migration and invasiveness and functional experiments demonstrated that the knockdown of S100A1 reversed the effects induced by FOXD2‘AS1 Furthermore S100A1 knockdown suppressed breast cancer progression by inhibiting the proliferation migration and invasiveness of McF7 and MDA‘MB‘ cells In agreement with these findings S100A1 has been reported to be overexpressed in ovarian cancer and to be associated with lymph mode metastasis the overexpression of S100A1 was shown to enhance cellular proliferation and migration whilst its inhibition exerted an opposite effect on ovarian cancer cells Moreover high tumor expression levels of S100A1 have been shown to be positively associated with decreased relapse‘free survival time in an endometrioid subtype of ovarian and endometrial cancers It was thus hypothesized that S100A1 functions as an important regulator in breast cancer and may therefore be a promising therapeutic target for this as well as other types of gynecological cancerThe Hippo pathway is an important signaling pathway that regulates cellular proliferation and apoptosis the activation of which is triggered by the phosphorylation of the large tumor suppressor kinases LATS1 and LATS2 The Hippo pathway is very complex as a number of kinases relay upstream signals to LATS to regulate this pathway The STE20 protein kinases MST12 as the core components of the Hippo pathway are considered responsible for the phosphorylation and activation of LATS12 YAP a downstream effector of the Hippo pathway is highly activated in various types of cancer and targeting YAP may effectively suppress tumorigenesis Both dysregulated Hippo signaling and aberrant YAP activation contribute to cancer progression In the present study FOXD2‘AS1 knockdown significantly increased the phosphorylation of YAP and LATS1 indicating that the Hippo signaling pathway was activated by FOXD2‘AS1 downregulation Notably it was found that MST12 expression levels were downregulated by
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Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen “ response as defined by ‰¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject –º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add –º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice –º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately “ of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access“ months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles “ weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles “ weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigator™s discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA “ measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA “ level by ‰¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ‰¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ‰¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the χ² test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA “ PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics “ years “ years ‰¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA “ decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had ˜exceptional™ responses defined as normalisation or ‰¥ decline of CA “ patients or normalisation PET SUVmax in In Arm B had ‰¥ CA “ decline at EOT response by RECIST PR ‰¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an œexceptional CA “ response and had an ˜exceptional™ PET response as defined by either ‰¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA “ response ‰¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection status“““““““““AAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA “ decrease FDG PET SUVmax decrease ‰¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons™ comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigator™s choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a ˜softening™ of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA “ PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA “ decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA “ response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA “ and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options
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the diagnostic platform utilizing the detection of biomarkers in various body fluids called œliquidbiopsy can revolutionize precision medicine precision medicine is aimed at attaining betterpersonalized care by the development of the latest diagnostic and prognostic methods thatconsider individual variability kaur liquid biopsy is being utilized for noninvasiveabbreviations 5hmc 5hydroxy methyl cytosine ccfdnas circulating cellfree deoxyribonucleic acids ccffetalnascirculating cellfree fetal nucleic acids ccfmirnas circulating cellfree mirnas ccfnas circulating cellfree nucleic acidsccfrnas circulating cellfree ribonucleic acids mtdna mitochondrial dnaedited byrui henriqueportuguese oncology instituteportugalreviewed bynaoko hattorinational cancer center researchinstitute japanigor kovalchukuniversity of lethbridge canadacorrespondencejyotdeep kaurjyotdeep2001yahoocoinspecialty sectionthis was submitted toepigenomics and epigeneticsa section of the frontiers in geneticsreceived february accepted july published august citationrahat b ali t sapehia dmahajan a and kaur j circulating cellfree nucleic acids asepigenetic biomarkers in precisionmedicine front genet 103389fgene202000844frontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersprognostic and predictive purposes efficient and reliable markerswithin the body fluids can help in personalized treatmentdecisions for monitoring disease and survival ccfnas haveemerged as such markers for screening diagnosis prognosismanagement and treatment of various cancers autoimmuneneurological and mitochondrial diseases prenatal diagnosisdiagnosis of pregnancyrelated complications pos diabetes ‚ammation rheumatoid arthritis stroke and traumaswarup and rajeswari an increased amount of ccfnasis observed in these disorders making liquid biopsies moresensitive rapid accurate and preferable alternatives for variousinvasive diagnostic methods pos ccfnas present in blood circulation include cellfree genomicdnas ccfdnas and cellfree mtdna kohler thierry and cellfree rnas ccfrnas includingproteincoding messenger rna mrna regulatory noncodingrnas like micrornas mirnaslong noncoding rnaslncrnas circular rnas and rnas involved in translationlike transfer rnas trnas and ribosomal rnas rrnaspos the ccfnas dnas and rnas are generally released into theblood circulation either by apoptosis necrosis or active secretionin healthy persons the origin of ccfnas is mainly attributed tolymphoid and myeloid tissues snyder while in thecase of various clinical conditions the associated or the aï¬ectedtissues would release the extra amount of ccfnas into bloodswarup and rajeswari devonshire in a patternspecific to the pathophysiological condition hunter noferesti various genetic as well as epigenetic biomarkers havebeen explored for ccfnabased liquid biopsy as geneticbiomarkers are less consistent and provide more variabilityacross studies epigenetic markers which are more generalizedbetween samples present as a promising alternative for earlydiagnosis and monitoring of the diseases these epigeneticmarks are tissue specific and reflect the pattern of diseaseprogression zeng furthermore epigeneticbiomarkers are dynamic with most techniques required foranalysis ofthese biomarkers that are already available inclinical laboratoriescare thein future precise patientthe use of epigenetic marks has revolutionized the fieldof noninvasive molecular diagnosisreplacing traditionalscreening and treatment methods these assays have greatpotentialepigeneticmarks for ccfnas reflect the pattern specific for the tissuecontributing to these ccfnas therefore the use of epigeneticmarkers can help in the diagnosis of various diseases evenbefore the onset of actual symptoms and hence help inbetter management ofthe disease precision medicine hasimproved health care by theidentification of diï¬erentstagessubsets of diseases precise diagnosis and treatmentfurthermore the development of advanced analytical softwaretechniques like machine learning and artificialintelligencecan enhance precision medicine ahlquist beltrangarcia these are used in combination withnextgeneration sequencing to identify novel ccfnabasedepigenetic markersepigenetic biomarkers in ccfnasreliable markers are required to guide personalized treatmentdecisions for monitoring disease progression and survivalthe presence of epigenetic marks on ccfnas specific toa particular clinical condition is widely being explored toadvance personalized medicine a perfect epigenetic markerfor precision medicine should be able to detect the diseasewith high sensitivity predict the risk of disease developmentand its progression and monitor the therapeutic responseofbeltrangarcia ccfdnas areassociated with various epigenetic marks schwarzenbach like dna methylation hydroxymethylcytosine 5hmcand posttranslational modifications of histones in additionnucleosome positioning and occupancy on ccfdnas haveexhibited high sensitivity and specificity in liquid biopsybasedmethods for disease detection and classificationthe patientthe5methylcytosine5mc modificationat cpgdinucleotides is the most abundant form of dna methylationit plays an important role in the regulation of gene expressionand is widely used as an epigenetic biomarker for ccfdnabasedassays dna methylation has replaced many genetic mutationor proteinbased markers these 5mc biomarkers are alsovaluable in identifying tissuespecific methylation to estimatetumor burden and tissue of origin in ccfdnas in additionto 5mc 5hydroxymethylcytosine 5hmc is also used as anepigenetic mark on ccfdnas zeng 5hmc is createdby the oxidation of 5mc by “ translocation tet proteinsalthough 5hmc is far less abundant compared to 5mc it ismore distinctly distributed among diï¬erent transcriptionallyactive regions which emphasizes its potential as a diagnosticmarker genomewide analysis of 5hmc pattern can providemore information about the potential of this epigenetic markerfor ccfdnas zeng nucleosome positioning has emerged as a recent biomarkerto distinguish the tissue of origin of ccfdna based onderived nucleosome maps snyder performed deepsequencing on ccfdnas and observed a distinct pattern ofnucleosome positioning between healthy persons and cancerpatients correlating with the tissues of origin snyder this emphasizes the use of nucleosome maps whichconsist of occupancy of transcription factor and nucleosomeas the epigenetic marks to distinguish normal versus cancerccfdnas hence nucleosome positioning can also be used toidentify various cancers that generally require invasive biopsiesfor definitive diagnosis moreover genomewide nucleosomepositioning of ccfdnas is utilized to infer pathological statesof multiple disease types a comprehensive public databasecalled cellfree epigenome atlas cfea provides the epigenomeprofile of ccfdnas from various human diseases and canhelp in a better understanding of collected data yu ccfdna are generally associated with nucleosomesand histone proteins histone proteins are posttranslationallymodified at amino acid residues located on their n andcterminal tails these modifications act as epigenetic marksthat can specifically distinguish diseaserelated ccfdnas in bloodsamples various types of histone modifications are associatedfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersarewith the development and pathogenesis of human diseaseszhao and shilatifard in addition to dna markers rna markers like mrnasmirnas lncrnas and circrnas are also getting attention in thefocus of clinical research pos most of the currently available diagnostic tests based onccfnas use either dna methylation markers or the diï¬erentialexpression of mirnas these biomarkersrelativelyeasily detected and estimated using accessible techniqueslike methylight methylspecific pcr methylationsensitivehighresolution melting and pyrosequencing garc­agimnez dna methylation specific to fetal and tumor dnahas been reported in pregnant women and cancer patientsrespectively wong poon the pattern ofthe methylation in these ccfdnas has been traced back to theirtissue of origin lun sun diï¬erentiallymethylated markers have been reported in ccfdnas like inspromoter in diabetes and reg1a and cux2 genes in pancreaticcancer lehmannwerman promoter methylationof serpinb5 rassf1a and stat5a act as epigenetic fetalmarkers in maternal blood chim chan rahat 2016atumor dna depending on the copy number mostly targetedmethylation sequencing is carried out in such cases which hasa greater potential for the detection of lower levels of ccfna inpatients with earlystage diseasechromatinbased chipseq experiments are revolutionizingour understanding of the complexes associated with chromatindynamics ongoing advances such as nanochipseq allow chipseq to be analyzed from far fewer cells necessary for embryologyand development studies nakato and shirahige theemergence of chipexo that digests the ends of dna fragmentsnot bound to protein is quite promising furey howeverthe application of these techniques to identify biomarkers islimited due to the expertise and cost associatedcriticalchipseq also providesinformation on otherchromatin modifiers such as histone marks and the enzymesthat modify these marks in diseases such as cancer chipseq has identified the role of aberrant h3k79 methylationby the methyltransferase dot1l in mixed lineage leukemiamllrearranged leukemias bernt in additionto chipseq diï¬erentlike chippcr elisabased assays or mass spectrometry are used to detect andquantify histone modifications on ccfnas in serum or plasmaadli and bernstein techniquesdiagnostic approach forepigenetic modifications in ccfnathe various diagnostic approaches to study the epigeneticmodifications in the nucleic acids include methylated cpgisland recovery assay mira and methylcap that rely onmethylcpgbinding domains mbd to capture methylateddna after dna fractionation either by restriction digestion orsonication mitchell these methods can also becombined with microarray or ngs technologies methylcapseq to identify biomarkers for cancer diagnosis and dnamethylation maps of cancer genomes simmer reduced representation bisulfite sequencing rrbs meissner is an efficient method for absolute quantification ofthe methylation status of more than one million cpg sites atsingle basepair resolution covering regions of moderate to highcpg density lee new techniques such as wholegenome bisulfite sequencing wgbs allows for an unbiasedassessment of dna methylation at singlebase resolution withfull coverage of more than million cpg sites in the humangenome and by using this technique in the clinical settingsrelevant biomarkers were identified in colorectal and breastcancers and certain types of leukemia berman some of the techniques are used in clinical settingslikeparallel shotgun sequencing and targeted sequencing norwitzand levy for noninvasive prenatal testing wgs for fetalgene detection lo and cancer personalized profilingby deep sequencing cappseq to quantify circulating tumordna newman despite the advancement of the techniques to study epigeneticmodifications the use of epigenetic biomarkers present onccfnas is limited due to their lower levels in the blood circulationin the case of cancer wgs is applied to only “ of cellfreeccfnas as epigenetic biomarkersin various diseasesthe detection and quantification of ccfnas viz rna dnafetal dna fetal rna mtdna and mitochondrial rna andmirna levels in body fluids are of clinical significance theseccfnas have the potential to act as biomarkers for diagnosisas well as prognosis of various diseases fleischhacker andschmidt breitbach such as diï¬erent cancersobstetric autoimmune neurological and mitochondrial diseasesas well as prenatal diagnosis etc kandel shaw although the most studied area of epigenetics is dnamethylation yet in the clinical setting there are only a fewmethylation markers various blood or tissuebased cohortwellpowered studies have recently shown that changes in thedna methylation are not only observed frequently in cancersbut also in other broad range of complex diseases includingneurodegenerative metabolic autoimmune and ‚ammatorydiseases although at a lower frequency tost dna methylation analysis of ccfdna might provide avaluable option in some cases when the blood“brain barrieris temporarily disrupted it was recently demonstrated by thedetection of unmethylated fragments of mbp3 and wm1 specificfor oligodendrocytes in about of patients with relapsingmultiple sclerosis zachariah cfrnas are also presentin the patient™s serumplasma in addition to ccfdnas higherlevels of circulatory rnases were observed in cancers and variousdiseases like cerebral attack preeclampsia etc and surprisinglyrna found in the circulation was found to be stable umu changes in the expression of intracellular mirnahave been causally linked with many diseases that include canceresquelakerscher and slack cardiovascular diseasesfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersnavickas neurodegenerative diseases gupta etc such changes in expression of mirna are eithersimilar or distinct in the serum of a particular set of patientsthus enabling mirna detection in serum as biomarkers ofhuman diseases backes therefore ccfnas playa prominent role in the pathogenesis and diagnosis of variousdiseases further research is required in this field to ensure thewidespread application of these markers in clinical settingsccfnas in cancerevery year about million new cases of cancer are reportedexcluding skin cancer other than melanoma that cause about million deaths accounting for of deaths in a yearferlay an estimated number of more than million new cancer cases are likely to be diagnosed and cancer deaths are expected in the united states in whichdeciphers almost deaths per day siegel thesix major hallmarks of cancer hanahan and weinberg are uncontrolled cell growth and division programmed cell deathavoidance invasion metastasis and angiogenesis the diagnosisof cancer usually occurs following the appearance of signs orsymptoms or through screening and investigations like xraysblood tests endoscopy ct scans etc biopsy tissue examinationindicates the type of proliferating cells genetic abnormalitiesand histological grade and other characteristics thereforeadvanced measures such as estimating prognosis risk assessmentfor early diagnosis biomarkers and observing the response totherapy can lead to successful treatment positive outcomes andimprovement of the quality of life for patients the tissue biopsymatched ccfdna is considered as surrogate marker due to itsrelease from the tumor sites de mattosarruda it is proven to be a noninvasive rapid and sensitive markerfor diagnosis prognosis and therapy response monitoring indiï¬erent cancers volik in addition the integrityof ccfdna extent of ccfdna fragmentation may be utilizedas a promising biomarker for diagnosis and prognosis of cancermadhavan ccfnas as diagnostic and prognosticbiomarkers for cancerserum or plasma ccfna serves as a œliquid biopsy which is usefulfor various applications in diagnostics and avoids the necessityfor biopsy of tumor tissue the levels of ccfna in blood andlymphatic circulation are aï¬ected by degradation clearance andvarious other physiological events liver and kidney clear nucleicacids from the blood and they have a halflife of diï¬erent timeintervals in the circulation that varies from min to severalhours fleischhacker and schmidt mirnas appear to beextremely stable but their rate of clearance from the blood is notwell studied in cancer patients thus owing to the uniqueness ofthis research areaccfdnas in cancerccfdnas consists of both genomic dna gdna as well asmtdna there is a production of longer uneven fragments ofdna by necrosis in cancer patients and shorter dna fragmentsfrom apoptosis hence increased levels of longer dna fragmentsin the bloodstream have been targeted as a potential marker forthe presence of malignant tumor dna arkoboham tumor cells are the origin of ccfdna in the blood of cancerpatients stroun aberrations specific to tumors likeoncogene and tumor suppressor gene mutations wang methylation of dna fujiwara and instabilityof microsatellite dna shaw were recognized inccfdna tumorigenesis and its progression are monitored bythe change in various epigenetic modifications patients withdiï¬erent types of malignancies have methylated dna in theirserum or plasma one of the most important methods foranalyzing malignancy is by detecting the presence of methylatedccfdna in cancer patientsfor early diagnosis of colorectal cancer crc analysisof promoter hypermethylation in blood and fecal dna hasthe potential to be used as a noninvasive test and eï¬ortsare made for clinical application of these molecular markersvarious studies have observed mgmt rassf2a wif1 ngfrand sept9 as aberrantly methylated genes used as diagnosticbiomarkers in patients with crc lee powrozek several potential methylation biomarkers have beenfound that diï¬erentiate plasma from breast cancer patients andthat from control subjects hoque remarkablytwo independent studies recognized cst6 as being methylateddiï¬erentially between breast cancer and control plasma samplesradpour chimonidou for lungcancer an early focus was to search methylated cdkn2a as aplasma diagnostic biomarker studies observed hypermethylationof cdkn2a in the plasma of patients with lung cancer ascompared to cancerfree controls zhang shox2was identified as a potential biomarker in a retrospective studydone by researchers from the theracode a diagnostic firmkneip a recent study by a group as part ofthe australian pancreatic cancer genome initiative apgihas observed elevated levels of aberrant methylation in theimportant cell signaling pathways thus suggesting its possibilityas a disease biomarker they worked on a group of sixcandidate genes nptx2 sarp2 uchl1 ppenk cdkn2aand rassf1a and observed diï¬erential methylation in thepromoters of all the genes in pancreatic cancer and healthycontrols except in cdkn2a promoter which was methylateddiï¬erentially between pancreatic cancer patients and thosehaving chronic pancreatitis park epigenetic eventsin the progression of cancer include the promoter regionthe genes piclass gstp1 and apchypermethylation ofwhich are the most common somatic genome abnormalities incolorectal and prostate cancer ellinger rassf1ararb sept9 esr1 and cdkn2a are the important methylatedgenes that have shown utility in prognosis using ccfdnaassays in many patients methylation of histones is an activeprocess with vital roles in diï¬erentiation and developmenttumorigenesis also occurs due to aberrant levels of histonemethylation the promoters associated with h3k4 are primarilytrimethylated by set1a and set1b set1a plays a vitalrole in oncogenic function in breast cancer metastasis lungfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerscancer and colorectal cancer zhao and shilatifard table presents the frequently hypermethylated genes invarious cancer typesccfmirnas in cancerin various cancers mirna expression dysregulation has beenobserved that suggests its role in many processes necessaryfor the progression of cancer like proliferation cell deathmetastasis and resistance to treatmentiorio and croce during the development of the liver mirna expressionchanges dynamically mir500 is one such oncofetal mirnathat is important for the diagnosis of hepatocellular carcinomayamamoto lately in nonsmall cell lung cancernsclc mir1246 and mir1290 were recognized as tumorinitiating and cellspecific mirnas zhang mir was found to be a significant prognostic factor for osccpatients based on cox regression analysis in addition mir could serve as a valuable biomarker in oscc patientsto predict the clinical response to chemoradiotherapy lin a study by alhasan showed a serumsignature of 5mirnas mir135a mir106a mir200c mir and mir433 predicted a very highrisk prostate canceralhasan expression levels of mir21 mir23bmir200c and mir200b were upregulated in metastatic breastcancer when compared to early breast cancer patients thereforesupporting the notion that ccfmirnas presents a tool with thecrucial diagnostic and prognostic implication in breast cancerpapadaki furthermore a study discovered thatincreased mir122 expression was significantly associated witha reduction in the overall survival as well as progressionfreesurvival in breast cancer patients saleh elevationin the levels of serum mir29 mir122 mir155 and mir was observed in cholangiocarcinoma although mirnaslevels before surgery were inappropriate as survival prognosticmarker however postsurgery decrease in the serum mir122levels was significantly linked with better patient prognosisloosen ccfnas in treatment and cancerprogressionccfdna analysis is a noninvasive process that allows day to daypatient followup and monitoring of response toward treatmentges both genetic and epigenetic changes areexhibited by ccfdna stroun the study of thesechanges might provide valuable information to mold the choiceof treatment by clinicians given the limitations of the noveltargeted therapiesabnormal hypermethylation at cpg islands occurs rarely innonmalignant and normally diï¬erentiated cells so the releaseof dna from tumor cells can be found with a prominentextent of sensitivity even when the excess of dna is releasedfrom normal cells and this characterizes its potential clinicalapplication wong in this context promoter regionhypermethylation of ink4a occurs very early in the progressionof hepatocellular carcinoma hcc and henceit serves asa valuable biomarker for noninvasive diagnosis as well asprediction of response to therapy huang isatherapyimmunotherapyidentification ofrapidly developingsignificant mirnasinmany cancers because of various advantages over standardchemotherapythatprovides a foresight of response in cancer immunotherapy wouldenable better patient selection and enhancement of therapeuticefficacy and provide a novel target antonia chen mirna21 is a cellfree oncogenic mirna whichhas been known as a potential regulator of stat3 and thusit could be detected in various tumors ji thuscirculating mirna21 can act as a biomarker for response incancer immunotherapy wu in the mycnamplified neuroblastoma progression mycnis detected in circulating dna this phenomenon was found tobe associated strongly with the quick progression of tumors andpoor outcomes combaret loss of heterozygosityloh and abnormal methylation at the promoter region ofmycn were detected using ccfdna which showed elevatedlevels in patients of highgrade glioma detection of promoterregion hypermethylation of myod1 in serum may serve asa potential prognostic marker for discriminating patients ofcervical cancer at high risk for lymph node metastasis or relapsewidschwendter moreover the investigation of circulating mirnas presentsgreat potentialin revealing new insights into their role intherapy and diagnosis mirna serum signatures mir345 5pmir330 3p and mir9 3p were found to be significantlyupregulated in patients of prostate cancer pca when comparedto healthy individuals the role of mir3455p to act as anoncomir through cdkn1a targeting makes it a potential targetfor pca therapeutically tinay ccfdnasin glioma wereassociated with diï¬erentialmethylation levels of mgmt cyclindependent kinase inhibitor2a multiple tumor suppressor p16ink4a p73 and retinoicacid receptor beta rarb balana weaver wakabayashi all these studies propose acrucial role of epigenetic marks in ccfnas in cancertargetedtherapy as well as pathogenesisccfnas in cancer precision medicineprecision oncology is an approach that includes the molecularprofiling of tumors to identify eï¬ective therapeutic strategiesa clinical research program initiated by the englander institutefor precision medicine eipm in uses wholeexomesequencing of metastatic and primary tumors to identifyindividualized therapeutic options and to help guide clinicaldecision making by prospective followup of patients rennert oncology is the obvious choice for heightening theimpact of precision medicine several targeted therapies havebeen developed that have shown profound benefits recentlynovel immunological approaches produced insightful responsessnyder in addition the identification of epigenetic biomarkers leadsto more precise disease prognosis especially in therapeutic areasthat are linked with a high degree of variability concerningsurvival van neste research carried out in severalcancers like glioblastoma reveals that levels of 5hmc are criticalin the regulation of genes having a crucial role in disease andfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerstable frequently hypermethylated genes in various cancer typesgenecancer typereferencesitih5 dkk3 brca1 erbeta apc gstp1 esrbrassf1ap16arf bax bcl2 cdh1 dapk ednrb eomes faddpcdh17 pou4f2sept9 hltf nell1 cea tac1vhlrbtmeff2 prdm13ost2 mgmtapc gstp1st6galnac3 znf660brca1 rassf1a rassf2ahtertp16ink4a timp3 thbs1breast cancerprostate canceresophageal liver and pancreasbladder cancerkloten cheuk vu liu house abern wang colorectal cancerkidney tumorsretinoblastomalung cancerrenal cell carcinomaprostateovarian cancerleptomeningeal carcinomatosis in csfgliomatham semaan ma ohtanifujita palmisano lee su hauser haldrup giannopoulou lonning bougel liu show that global reduction in 5hmc over the genome leads topoor clinical outcomes in these patients johnson epigenetic changes introduced common genetic mutations inan in vitro model of lung cancer vaz epigeneticbased diagnostics can detect early disease signals and thuscan provide possibilities for clinicalintervention before theprogression of symptomsthe detection of ccfnas could be exploited by targetedtherapies approved lately and eventually benefit the patientsscrutinizing cancers by analyzing ccfna dynamics in blood orserum is an innovative and emerging research area as far as theexisting research advancement and the growth of the medicalindustry are concerned we consider that ccfna assays may beemployed for realtime personalized treatments in the future forcancer patients based on their ccfnas or ccfdna methylationlevels for diagnosis and prognosis nevertheless there is muchscope for improvement before the application of this technologyin clinical settingsuse of ccffetalnas in prenataldiagnosis and pregnancyrelateddisordersthe apoptosisnecrosis ofduring pregnancytrophoblastsarising from syncytiotrophoblast is the prime source of therelease of ccffetalnasinto the maternal blood litton the presence of ccffetalnas has pavedthe way for noninvasive prenatal diagnosisand earlylo prediction of pregnancyrelated complications the use of ccffetalnas has gradually replacedinvasive techniques like amniocentesis or chorionic villussampling serr ccffetaldna comprises “ ofthe maternal ccfdna wang andcan be efficiently detected atthe fifth week of gestationguibert the amount of ccffetaldna inmaternal blood increases progressively throughout pregnancybirch ccfnas in prenatal diagnosisprenatal diagnosis is an established practice for the managementof pregnancy as well as avoidance of prenatalneonatal deathsthe leading causes for such deaths are genetic disorderbirth defects congenital malformations and chromosomalabnormalities like trisomy down™s syndrome edward™ssyndrome and patau syndrome and sex chromosomeaneuploidies like monosomy x turner syndrome carlson andvora therefore successful management of pregnancydemands efficient and timely prenatal diagnosis to determine theoutcome of pregnancy timely detection of neural tube defectsis already providing early prenatal treatment resulting in betterneonatal outcomes adzick ccffetaldna is clinically used for the detection of fetal sexand multiple anomalies based on paternally inherited mutationsbianchi recent studies have discovered many fetalepigenetic biomarkers for ccffetalnabased liquid biopsies inclinical samples that have demonstrated high clinical potentialin disease diagnosis prognosis and pregnancy managementthese epigenetic modifications are specific to the fetus and helpto distinguish fetal nucleic acids from maternal nucleic acidsjones and takai clinical testing of recently developedfetal epigenetic markers can help in the proper managementof personalized care the first reported use of fetalderivedepigenetic marker in maternal body fluids had come frompoon who utilized an imprinted h19igf2 locusbased on parentoforiginspecific methylation and the maternaland the paternal copies of the gene were distinguished inmaternal blood poon based on the placental originof ccffetaldna having placentaspecific dna methylationpattern the genomic regions that show diï¬erential methylationbetween the placenta and the maternal blood cells can actas a marker for fetal dna in maternal blood the promoterregion of maspin serpinb5 is the first such reported universalfetal dna marker with detectable hypomethylationin thebackground of hypermethylated maternal sequences the fetalorigin of these hypomethylated maspin has been confirmedby the clearance of these sequences within h of deliveryfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerschim the clinical use of hypomethylated maspinis limited by the required bisulfite treatment of ccffetaldna as this treatment can degrade around of the dnagrunau thus decreasing the amount of alreadylow levels of fetal dna in maternal blood such limitationwas overcome by the detection of fetalderived hypermethylatedrassf1a in maternal blood for prenatal diagnosis chan hyland tounta 2011b the maternalhypomethylated rassf1a ccfdna can be removed by treatmentwith methylationsensitive restriction enzyme digestion leavingbehind fetal hypermethylated rassf1a ccffetaldna chan various other fetalderived diï¬erentially methylatedsequences have also shown a similar potential to act as fetal dnaepigenetic markers in maternal blood table ccffetaldna methylation markers have the potential ofbeing used as both quantitative as well as qualitative markersin prenatal diagnosis as qualitative markers these are used toestimate the false positives during the determination of fetalgender rh status and paternally inherited polymorphisms chan while as quantitative markers these can estimate thelevels of ccffetaldna in maternal plasma such an applicationof ccffetaldna finds its use in the detection of chromosomalaneuploidies lun based on the location of themaspin gene on chromosome hypomethylated fetal maspinhas been used to calculate the allelic ratio to diagnose trisomy with sensitivity tong fetal trisomy was detected by analyzing chromosomal dosage via targetingof fetal hypermethylated hlcs sequences in the combinationof microfluidics digital pcr rassf1a on chromosome and zfy on the y chromosome were used as referencestong fragmentation pattern of ccffetaldnain maternal plasma has been successfully used for enrichmentmethod in size separation manner on agarose gel electrophoresisramezanzadeh various nextgeneration sequencing and highthroughputtechniques have catalyzed the identification of newer and novelfetal epigenetic markers further advancing noninvasive prenataldiagnosis the microarraybased approach has identified manyfetal epigenetic markers with diï¬erential methylation betweenchorionic villus samples and maternal blood on chromosomes and for aneuploidy detection chu combining highresolution tiling oligonucleotide array withmethylated dna immunoprecipitation medip has helped ina genomewide screen for detecting the diï¬erential methylatedsites between placental tissue and maternal blood cells it hasdetected various new fetal epigenetic markers on chromosomes and
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“linkinghubelseviercomretrievepiis0968000400015954 101016s0968000400015954 wang y huang s sah vp ross j brown jh han j cardiac muscle cell hypertrophy and apoptosis induced by distinct membersof the p38 mitogenactivated protein kinase family j biol chem “ 101074jbc27342161 saurin at martin jl heads rj foley c mockridge jw wright mj the role of differential activation of p38mitogenactivatedprotein kinase in preconditioned ventricular myocytes faseb j “ 101096fj990671com podewski ek hilfikerkleiner d hilfiker a morawietz h lichtenberg a wollert kc alterations in janus kinase jaksignaltransducers and activators of transcription stat signaling in patients with endstage dilated cardiomyopathy circulation “10116101cir000005754582749ff patel ab and verma a covid19 and angiotensinconverting enzyme inhibitors and angiotensin receptor blockers what is the evidencejama “ jamanet
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" adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ‰¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically œcold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12“ these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence “ to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments dose“response and time“course were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23“ and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturer™s protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at “ cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering × ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as × compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17“ thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3a“dad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once × or twice × to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n4“ micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 a“d balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only × pre exposure and × pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5b“d similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5e“g as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5e“gdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans a“c to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive × and × ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations a“g balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot e“g g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with œno evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw
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chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis 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 cited for commercial reuse please contact spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ˆ†ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewer™s yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period “ d under the short photoperiod the diapause maintenance period “ d under the short photoperiod the diapause termination period “ d under the short photoperiod and the reproduction period “ d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about “ a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about “ a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at ˆ’°c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at ˆ’°c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content “ annealing temperature “°c and primers length “ a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 ˆ’slopeˆ’ the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0× chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the “°c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ˆ†ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ˆ†ct method the stability of genes was ranked according to the sd of genes ˆ†ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ˆ†ˆ†ct method the differences of target genes™ expression levels were analyzed by tukey™s test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 “ rps26e “ and actin “ followed by 128up “ arp3 “ arpc5 “ rps5 “ and ef1a “ the genes with lower expression abundance were αtub “ and gapdh “ according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ˆ†ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ˆ†ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ˆ†ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ˆ†ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ˆ†ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ˆ†ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ˆ†ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ˆ†ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ˆ†ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ˆ†ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ˆ†ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ˆ†ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ˆ†ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ˆ†ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ˆ†ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ˆ†ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ˆ†ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o
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to machine learning with python a guide for data scientists o™reilly media inc california demÅ¡ar j statistical comparisons of classifiers over multiple data sets j mach learn res “ yates a the ensembl rest api ensembl data for any language bioinformatics “ kim e r chang d k colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis world j gastroenterol diovasc dis “ schulte d small dense ldl cholesterol in human subjects with different chronic inflammatory diseases nutr metab car smedley d biomartbiological queries made easy bmc genom quinlan a r hall i m bedtools a flexible suite of utilities for comparing genomic features bioinformatics “ 101093bioin forma ticsbtq03 rom¡n j evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis inflamm bowel dis “ 101002ibd23020 song r identification and analysis of key genes associated with ulcerative colitis based on dna microarray data medicine baltimore e10658 101097md00000 schwegmann k detection of early murine colorectal cancer by mmp29guided fluorescence endoscopy inflamm bowel dis “ 101097mib00000 oliveira l g d positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis arq gastroenterol “ 101590s0004 shin js antiinflammatory effect of a standardized triterpenoidrich fraction isolated from rubus coreanus on dextran sodium sulfateinduced acute colitis in mice and lpsinduced macrophages j ethnopharmacol 158pt a “ 101016jjep201410044 owens d w lane e b keratin mutations and intestinal pathology j pathol “ 101002path1646 macfie t s duox2 and duoxa2 form the predominant enzyme system capable of producing the reactive oxygen species h2o2 in active ulcerative colitis and are modulated by 5aminosalicylic acid inflamm bowel dis “ 10109701mib00004 0e palmer n p concordance between gene expression in peripheral whole blood and colonic tissue in children with inflammatory bowel disease one e0222952 101371journ alpone02229 wei z large sample size wide variant spectrum and advanced machinelearning technique boost risk prediction for inflammatory bowel disease am j hum genet “ amrhein v greenland s mcshane b scientists rise up against statistical significance nature “ wasserstein r a0l schirm a a0l lazar n a0a moving to a world beyond œp  maeda y fully automated diagnostic system with artificial intelligence using endocytoscopy to identify the presence of histologic inflammation associated with ulcerative colitis with video gastrointest endosc “ 101016jgie201809024 acknowledgementsthis research was partially supported by grants from the natural sciences and engineering research council of canada nserc to hu grant number rgpin and to lpc grant number rgpin hmk was partially supported by funding from memorial university™s school of graduate studiesauthor a0contributionsconceptualization hu and lpc methodology hmk hu and lpc analysis hmk and lpc writing hmk hu and lpc experiments hmk supervision hu and lpccompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to hu a0or a0lpcreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020705830vol1234567890wwwnaturecomscientificreports 0copen 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 license and indicate if changes 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 if material 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 obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020705830vol0123456789wwwnaturecomscientificreports 0c'
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"To assess DFS the disease status was monitored every 2 months for the first 6 months and subsequently every 3 months by computed tomography after enrollment and according to good medical practice. Toxicities related to the administration of chemotherapy were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0; ctep.cancer.gov). DFS was defined as the time from the date of enrollment to disease recurrence or death due to any cause and estimated according to the Kaplan-Meier method. A Cox regression model was fit with the time from surgery to enrollment as a covariate to evaluate its effect on DFS. A natural log transformation was applied to the raw protein measurement data and the Pearson correlation coefficient was used to test associations. Bivariate comparison of baseline characteristics between the assigned treatment groups was performed using the Fisher exact test for categorical variables or the Student t test or Wilcoxon rank sum test for continuous variables. A multivariable logistic model to evaluate baseline factors and treatment assignment was fit using backwards selection. Median ERCC1 and RRM1 expression levels were compared with historical medians using the 1-sample Wilcoxon signed rank test. The percentage of patients with both ERCC1 ??65 and RRM1 ??40 was compared with the historical rate using a chi-square test. All statistical analyses and graphics were performed using SAS statistical software (version 9.2; SAS Institute Inc Cary NC). A significance level of 5% was used for all analyses. RESULTS Patient and Trial Characteristics To ensure an adequate sample size of eligible patients and biomarker-specific subgroups a total of 85 patients was registered between April 2 2009 and April 1 2011 from 27 participating sites. Four patients were ineligible; 3 had inadequate lymph node sampling and 1 did not have a tumor measuring ??2 cm. provides the characteristics of the 81 eligible patients. Patient Demographics and Disease Characteristics Variablesa All Patients Assigned to Chemotherapy Assigned to Observation P Refused Assignment Accepted Assignment P N = 81 N = 63 N = 18 N = 20 N = 61 Age y .37 .39 ?Median 64 63.3 68.8 67.2 63.3 ?Mean 63.5 62.9 65.5 65.2 62.9 ?Range 41.6“84.2 41.6“84.2 41.6“81.7 44.2“82.9 41.6“84.2 Sex .18 .61 ?Female 44 (54%) 37 (59%) 7 (39%) 12 (60%) 32 (52%) ?Male 37 (46%) 26 (41%) 11 (61%) 8 (40%) 29 (48%) Ethnicity .65 .18 ?Unknown 7 (8%) 5 (8%) 2 (11%) 0 (0%) 7 (11%) ?Non-Hispanic 74 (91%) 58 (92%) 16 (89%) 20 (100%) 54 (89%) Race .73b .75b ?African American 8 (10%) 8 (13%) 0 (0%) 2 (10%) 6 (10%) ?Asian 3 (4%) 2 (3%) 1 (6%) 0 (0%) 3 (5%) ?Pacific Islander 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) ?White 66 (81%) 52 (83%) 14 (78%) 17 (85%) 49 (80%) ?Unspecified 2 (2%) 0 (0%) 2 (11%) 1 (5%) 1 (2%) Histology .06c .60c ?Adeno 52 (64%) 44 (70%) 8 (44%) 14 (70%) 38 (62%) ?Squamous 25 (31%) 17 (27%) 8 (44%) 6 (30%) 19 (31%) ?Large 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Bronchioloalveolar 1 (1%) 0 (0%) 1 (6%) 0 (0%) 1 (2%) ?Other 2 (2%) 1 (2%) 1 (6%) 0 (0%) 2 (3%) Stage of disease .16 .27 ?IA (<3 cm) 25 (31%) 22 (35%) 3 (17%) 4 (20%) 21 (34%) ?IB (?3 cm) 56 (69%) 41 (65%) 15 (83%) 16 (80%) 40 (66%) Zubrod performance status .11 1.00 ?0 44 (54%) 31 (49%) 13 (72%) 11 (55%) 33 (54%) ?1 37 (46%) 32 (51%) 5 (28%) 9 (45%) 28 (46%) Weight loss (6 mo) 1.00d .31d ?<5% 64 (79%) 49 (78%) 15 (83%) 14 (70%) 50 (82%) ?5-<10% 9 (11%) 7 (11%) 2 (11%) 3 (15%) 6 (10%) ?10“20% 4 (5%) 3 (5%) 1 (6%) 2 (10%) 2 (3%) ?>20% 1 (1%) 1 (2%) 0 (0%) 0 (0%) 1 (2%) ?Unknown 3 (4%) 3 (5%) 0 (0%) 1 (5%) 2 (3%) Smoking status ?Current 33 (41%) 26 (41%) 7 (39%) 8 (40%) 25 (41%) ?Former (quit ?1 y) 39 (48%) 30 (48%) 9 (50%) 10 (50%) 29 (48%) ?Never 9 (11%) 7 (11%) 2 (11%) 1.00e 2 (10%) 7 (11%) 1.00e Abbreviation: Adeno adenocarcinoma. a All P values shown are 2-sided. b White versus all other races. c Adenocarcinoma versus all other histologies. d Weight loss <5% versus ?5%. e Derived using the Freeman-Halton exact test. The distribution of assignment to chemotherapy and observation was 63 patients (78%) and 18 patients (22%) respectively which was not significantly different (P?=?.20 Fisher exact test) from the expected rates of 70% (129 patients) and 30% (55 patients) respectively.16 Based on protein levels in these 81 patients the number of those with low ERCC1 and low RRM1 was 31 patients (38%) 22 patients had low ERCC1 and high RRM1 (27%) 10 patients had high ERCC1 and low RRM1 (12%) and 18 patients had high ERCC1 and RRM1 (22%) which is not significantly different from prior results (P?=?.14 Fisher exact test; 54 of 184 29%; 38 of 184 21%; 37 of 184 20%; and 55 of 1840.3 respectively). We investigated whether treatment arm assignment varied by patients' smoking status histology age and sex. In bivariate comparisons no statistically significant associations were found. However the multivariable logistic model found that patients with adenocarcinoma (P?=?.03) and potentially stage IA disease (P?=?.06) were more likely to be assigned to adjuvant chemotherapy (ie they were more likely to have low levels of ERCC1 RRM1 or both). One of the 18 patients assigned to observation and 19 of the 63 patients assigned to chemotherapy rejected this choice and withdrew consent. There was no statistically significant difference in patient characteristics between those who accepted and those who refused their treatment assignment (). Feasibility The trial achieved its primary feasibility objective with a treatment assignment within the prespecified timeframe in 71 of 81 patients (88%)."
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expanding cancer predisposition genes with ultra‘rare cancer‘exclusive 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 individual™s 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 Meier“Kaplan survival rate estimate b Meier“Kaplan diseaseprogressionfree estimatesomatic mutations in any of these genes is much longer a0months Applying the Kaplan“Meier survival estimate yields a p value of 178eˆ’ in the Logrank test Fig a04a The Kaplan“Meier diseaseprogressionfree estimate was also worse for patients with somatic mutations in the novel CPGs with a p value of 603eˆ’ 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 a0S1“S24 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 ˜Caucasians™”individuals that were both genetically verified as Caucasians and declared themselves as ˜white™ ˜nonCaucasians™”individuals 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 cancer”insights 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 world™s 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
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"One limitation of this study is the small number of cases under study although 15 FISH-positive cases is comparable to most other studies. The relatively small number of FISH-negative cases may have affected our ability to identify FISH-negative IHC-positive cases. However the study design does permit an assessment of the sensitivity of the IHC assay which is the most important consideration for a possible screening test. Our comparison of the immunohistochemical assays was not directly equivalent as the D5F3 assay included a proprietary tyramide signal amplification step whereas the ALK1 and 5A4 assays were conducted using our routine diagnostic detection system. However our study design also has several strengths. In particular the use of archival diagnostic paraffin blocks and FISH testing conducted in the course of routine diagnosis make the results of the study directly relevant to clinical practice. In summary we find IHC to be a highly sensitive (86%) and specific (100%) test for ALK rearrangement in lung adenocarcinoma. We find a slight advantage of a proprietary amplified assay (D5F3 Ventana) over two other antibodies with conventional DAB staining (ALK1 Dako and 5A4 Abcam) but only in scanty samples. Intensity of staining was the most discriminating measure and the proportion of cells staining did not contribute. We identified two cases that were positive for the ALK rearrangement by FISH but negative by all immunohistochemical assays and suggest that in discordant cases the IHC test result may be more predictive of treatment response than FISH. Further discordant cases need to be examined to help guide the treatment of these cases. Immunohistochemical testing is clearly at least a useful adjunct to FISH and we feel that it is reasonable in routine practice to use a sensitive IHC assay as a screening test. The danger of missing treatable cases using this method (i.e. FISH-positive IHC-negative crizotinib-sensitive tumors) appears very small especially when specimens contain adequate material. In difficult cases further investigations such as re-biopsy and repeated IHC/FISH may be helpful. Disclosure: This project was supported by the National Institute of Health Research Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London and the NIHR RM/ICR Biomedical Research Center. All other authors declare no conflict of interest. REFERENCES 1. Inamura K Takeuchi K Togashi Y EML4-ALK lung cancers are characterized by rare other mutations a TTF-1 cell lineage an acinar histology and young onset. Mod Pathol 2009 22 508 515 19234440 2. Koivunen JP Mermel C Zejnullahu K EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008 14 4275 4283 18594010 3. Shinmura K Kageyama S Tao H EML4-ALK fusion transcripts but no NPM- TPM3- CLTC- ATIC- or TFG-ALK fusion transcripts in non-small cell lung carcinomas. Lung Cancer 2008 61 163 169 18242762 4. Soda M Choi YL Enomoto M Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007 448 561 566 17625570 5. To KF Tong JH Yeung KS Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4-ALK variant. J Thorac Oncol 2013 8 883 891 23625156 6. McDermott U Iafrate AJ Gray NS Genomic alterations of anaplastic lymphoma kinase may sensitize tumors to anaplastic lymphoma kinase inhibitors. Cancer Res 2008 68 3389 3395 18451166 7. Kwak EL Bang YJ Camidge DR Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010 363 1693 1703 20979469 8. Lindeman NI Cagle PT Beasley MB Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists International Association for the Study of Lung Cancer and Association for Molecular Pathology. J Thorac Oncol 2013 8 823 859 23552377 9. Murakami Y Mitsudomi T Yatabe Y A Screening Method for the ALK Fusion Gene in NSCLC. Front Oncol 2012 2 24 22655265 10. Rodig SJ Mino-Kenudson M Dacic S Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res 2009 15 5216 5223 19671850 11. Peled N Palmer G Hirsch FR Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small-cell lung cancer. J Thorac Oncol 2012 7 e14 e16 22895149 12. Eisenhauer EA Therasse P Bogaerts J New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009 45 228 247 19097774 13. Selinger CI Rogers TM Russell PA Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol 2013 26 1545 1553 23743928 14. Conklin CM Craddock KJ Have C Laskin J Couture C Ionescu DN Immunohistochemistry is a reliable screening tool for identification of ALK rearrangement in non-small-cell lung carcinoma and is antibody dependent. J Thorac Oncol 2013 8 45 51 23196275 15. Sholl LM Weremowicz S Gray SW Combined use of ALK immunohistochemistry and FISH for optimal detection of ALK-rearranged lung adenocarcinomas. J Thorac Oncol 2013 8 322 328 23407557 16. Savic S Bode B Diebold J Detection of ALK-positive non-small-cell lung cancers on cytological specimens: high accuracy of immunocytochemistry with the 5A4 clone. J Thorac Oncol 2013 8 1004 1011 23689429 Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24292447 3915116 bjc2013735 10.1038/bjc.2013.735 Clinical Study A phase II multicentre study of ziv-aflibercept in combination with cisplatin and pemetrexed in patients with previously untreated advanced/metastatic non-squamous non-small cell lung cancer Ziv-aflibercept/cisplatin/pemetrexed in NSCLC Chen H 1 * Modiano M R 2 Neal J W 3 Brahmer J R 4 Rigas J R 5 Jotte R M 6 Leighl N B 7 Riess J W 3 Kuo C J 3 Liu L 8 Gao B 8 DiCioccio A T 8 Adjei A A 1 Wakelee H A 3 1Department of Medicine Roswell Park Cancer Institute Elm & Carlton Streets Buffalo NY 14263 USA 2Arizona Oncology/Arizona Clinical Research Center 1620W. St Mary's Rd Tucson AZ 85745 USA 3Department of Medicine Stanford University School of Medicine and Cancer Institute 875 Blake Wilbur Dr Stanford CA 94305 USA 4Department of Oncology The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins Bunting/Blaustein CRB 1650 Orleans St. G94 Baltimore MD 21231 USA 5Department of Medicine Norris Cotton Cancer Center Geisel School of Medicine at Dartmouth 1 Medical Center Drive Lebanon NH 03756 USA 6Rocky Mountain Cancer Centers 1800 Williams Street Suite 200 Denver CO 80218 USA 7Department of Medicine Princess Margaret Hospital and University of Toronto 610 University Avenue Toronto ON M5G 2M9 Canada 8Regeneron Pharmaceuticals Inc. 777 Old Saw Mill River Road Tarrytown NY 10591 USA *E-mail: hongbin.chenroswellpark. 04 02 2014 28 11 2013 110 3 602 608 29 08 2013 27 10 2013 30 10 2013 Copyright 2014 Cancer Research UK 2014 Cancer Research UK From twelve months after its original publication this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-sa/3.0/ Background: This study evaluated the efficacy and safety of ziv-aflibercept in combination with cisplatin and pemetrexed in non-small cell lung cancer (NSCLC). Methods: This single arm multicentre phase II trial enrolled patients with previously untreated locally advanced or metastatic non-squamous NSCLC. Patients received intravenous ziv-aflibercept 6?mg?kg?1 pemetrexed 500?mg?m?2 and cisplatin 75?mg?m?2 every 21 days for up to six cycles. Maintenance administration of ziv-aflibercept was to continue until disease progression intolerable toxicity or other cause for withdrawal. The co-primary end points were objective response rate (ORR) and progression-free survival (PFS). Planned sample size was 72 patients. Results: The study was closed prematurely because of three confirmed and two suspected cases of reversible posterior leukoencephalopathy syndrome (RPLS). A total of 42 patients were enrolled. Median age was 61.5 years;"
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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 stress3“current 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 risk15“clinical 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 studies30“given 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 œhigh œlow or œunclearliterature 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 œmeta 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 cochrane™s 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 japankoreakoreakoreakoreakoreakoreakoreakoreakoreaaustriaturkey““““““““““age 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 totalˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšnotes œ_ high risk œˆš low risk œ unclear riskˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšˆšsubmit 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 s1“discussionthis 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 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antibioticresistant pathogen strainlevel investigations are beginning to reveal themolecular mechanisms used by vrefm to colonize regions of the human bowelhowever the role of commensal bacteria during vrefm colonizationin particularfollowing antibiotic treatment remains largely unknown we employed amplicon16s rrna gene sequencing and metabolomics in a murine model system to try andinvestigate functional roles of the gut microbiome during vrefm colonization firstorder taxonomic shifts between bacteroidetes and tenericutes within the gut microbial community composition were detected both in response to pretreatment usingceftriaxone and to subsequent vrefm challenge using neural networking approaches to find cooccurrence profiles of bacteria and metabolites we detected keymetabolome features associated with butyric acid during and after vrefm colonization these metabolite features were associated with bacteroides indicative of a transition toward a preantibiotic naive microbiome this study shows the impacts of antibiotics on the gut ecosystem and the progression of the microbiome in responseto colonization with vrefm our results offer insights toward identifying potentialnonantibiotic alternatives to eliminate vrefm through metabolic reengineering topreferentially select for bacteroidesimportance this study demonstrates the importance and power of linking bacterial composition profiling with metabolomics to find the interactions betweencommensal gut bacteria and a specific pathogen knowledge from this researchwillinform gut microbiome engineering strategies with the aim of translatingobservations from animal models to humanrelevant therapeutic applicationscitation mu a carter gp li l isles ns vrbanacaf morton jt jarmusch ak de souza dpnarayana vk kanojia k nijagal b mcconvillemj knight r howden bp stinear tp microbemetabolite associations linked to therebounding murine gut microbiomepostcolonization with vancomycinresistantenterococcus faecium msystems 5e0045220101128msystems0045220editor manuel liebeke max planck institutefor marine microbiologycopyright mu this is an openaccess distributed under the terms ofthe creative commons attribution international licenseaddress correspondence to andre muandremuunimelbeduaureceived may accepted july published august julyaugust volume issue e0045220msystemsasm 0cmu keywords microbiome multiomics metagenomics metabolomics gutmicrobiome vancomycinresistant enterococci colonization antimicrobial resistanceceftriaxonevancomycinresistant enterococcus faecium vrefm is a significant health careassociated pathogen vrefm infections can be difficult to treat due to their intrinsicand acquired resistance to nearly all classes of antibiotics the world healthanization categorizes vrefm as a œhigh priority bacterial pathogen advocatingresearch to stop the global increase in antibiotic resistance recent studies highlightthe importance of the gut microbiota in modulating the growth and virulence of vrefmin the gastrointestinal ecosystem for instance the depletion of normal gut flora usingantibiotics exacerbates the severity of vrefm infection whereas transplant ofcommensal species including a consortium of clostridium bolteae blautia productablautia sartorii and parabacteroides distasonis can drive established vrefm colonization to below levels of culture detection specifically b producta”a colonizer ofthe colon”reduces vrefm growth in vivo by secreting a lantibiotic these observations raise the intriguing possibility that metabolic traits act in concert betweenpathogen and select gut commensals to confer mutual benefits during pathogenpersistence these findings also highlight the greater risk posed to immunocompromised patients when colonized with vrefm for instance allogeneic hematopoietic celltransplantation patients have gastrointestinal tracts that are dominated by vrefm as aresult of losing a large portion of the intestinal commensal microbiota upon receivingbroadspectrum antibiotics as pretreatment hildebrand discovered longtermecological impacts to the gut microbiome with strong bacterial species turnover afterceftriaxone treatment in humans further mice receiving broadspectrum antibioticscombination of metronidazole neomycin and vancomycin showed markedly increased vrefm colonization of the cecum and colon the compromised intestinal innateimmune defenses in these animals allowed proliferation of vrefm caused by theantibiotic exposure and subsequently reduced the expression of antimicrobial molecules produced by bacteria in the intestinal mucosa the problem with vrefm is further complicated by the fact that enterococci aremembers of the gastrointestinal tract microbiota a key reservoir of antimicrobialresistance amr genes and potentially facilitating gene transfer within the gut microbiome for example the vanb resistance gene was detected in human fecalspecimens that did not contain culturable vre and instead demonstrated that isolatescarrying the resistance transposon are anaerobic commensal bacteria eggerthella lentaand clostridium innocuum colonization of and persistence in the gastrointestinaltract therefore presents as a key mechanism for de novo vre and may lead to severeinvasive diseasethe current study aimed to understand the impact of antibiotics on the murine gutmicrobiota and the subsequent colonization pattern of vrefm to this extent wedesigned a murine model timeseries study that consisted of two main perturbativephases i antibiotic pretreatment with ceftriaxone and ii vrefm challenge our 16srrna gene profiling analyses highlighted a firstorder shift in bacterial biodiversitycomposition across time a secondorder clustering of samples associated with theexperimental phases and the transition of the postvrefm colonization gut microbiotaand its metabolome toward resembling an asymptomatic carriagelike microbiomephenotype this research provides support for engineering the metabolic potential ofthe gut microbiome using for example prebiotics as a nonantibiotic alternative fortreating multidrugresistant bacterial infectionsresultsexperimental design the following experimental design was developed to address the hypothesis that there are specific murine gut microbiome factors thatfacilitate vrefm colonization three groups of three c57bl6 mice cocaged wildtypejulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vretable summary of samples analyzed in this studyday of exptphase of exptannnnnnabxtxabxtxabxwnvreevreevreevreevrelvrelamplicon 16s rrnagene databœ“œ“œ“«º«ºœ“œ“œ“œ“œ“«º«ºœ“œ“œ“metabolomicsbœ“«º«º«º«ºœ“œ“œ“œ“œ“«º«º«º«ºœ“avg no ofobserved sotusc«º«º«º«ºathe key phases of the experiment where n represents naive abxtx represents antibiotic treatment abxwn represents antibiotic weaning vree represents earlyphase postvrefm colonization and vrelrepresents latephase postvrefm colonizationbsymbols œ“ sample processed «º data unavailablecthe average number of sotus observed across all mice for each day of the experimentmales were monitored and fecal samples were collected over a 14day period with twointervention time points including i ceftriaxone treatment administered at 05gliter indrinking water across a 2day period and ii colonization via oral gavage with «» vrefm st796 per mouse postantibiotic treatment at a single time point mice werehoused in groups of five and samples were collected from the same three mice torepresent technical replicates per cage herein each group of cohoused mice will bereferred to as group a group b and group c the remaining two mice per group werereserved for microbiological assays table highlights samples and data sets collectedamplicon 16s rrna gene sequencing revealed firstorder shifts in bacterialcommunity composition amplicon 16s rrna gene sequencing was performed tocapture the bacterial community composition in an effort to track changes in responseto antibiotic pretreatment and vrefm colonization bacterial community profiles wereassessed in fecal samples from nine mice before during and after the two interventionstable a total of of reads reads passed quality control with reads on average per sample and a total of exact variant sequence typesie features with an average of features per sample and an upper bound of features when rarefied to reads alpha rarefaction analysis demonstratedsufficient sequencing depth to capture microbial diversity to saturation see fig s1 inthe supplemental materialthe biodiversity profiles of each sample were compared and showed that keysuboperational taxonomic units sotus were differentially abundant throughout thecourse of the experiment fig there was a shift in the dominance of bacteroidiabacteroidetes light green colored bars during the naive phase of the experiment tomollicutes tenericutes fuschia colored bars in response to ceftriaxone treatment witha return to the predominance of bacteroidia during the late phase of the experimentafter vrefm colonization ie days to of note is the predominance of lactobacillales in mouse to from group a fig the murine gut microbiota responds to antibiotics and microbial communityrichness begins to rebound days after vre colonization principalcoordinateanalysis pcoa of the unweighted unifrac distances was used to assess clusteringof fecal samples based on bacterial composition this assessment showed that the fecalmicrobiota from samples collected from each phase clustered together but were clearlyseparated between phases after exposure to ceftriaxone and challenge with vrefmpostantibiotic treatment fig 2a permutationbased statistical testing demonstratesthe groups are significantly different from one another fig s2 temporal tracking ofjulyaugust volume issue e0045220msystemsasm 0cmu fig biodiversity plot of sotus as relative frequencies at the taxonomic level of class firstorder shifts in microbial communitycomposition as revealed by 16s rrna gene community profiling from a predominance of bacteroidetes to tenericutes and return tobacteroidetes was observed each column displays the relative bacterial community composition in a mouse fecal sample collecteddaily and sorted by the chronology of the experiment ie day of experiment table the columns are further sorted by group iegroup a group b and group c and individual mice within each group mouse mouse and mouse stacked bars are presentedas relative frequencies at the taxonomical level of class however annotations of key taxa are at the phylum level bacteroidetes [green]firmicutes [gray] and tenericutes [fuschia] or order level lactobacillales [yellow]the changing microbiomes against each mouse on the pcoa sample space demonstrated a clear unidirectional trajectory that followed the chronology of the experiment106084m9figshare12775859 procrustes analyses of weighted andunweighted unifrac distances showed that the same general patterns on the samplespace were preserved meaning that there is congruency in global spatial patternsbetween qualitative and quantitative measures of community dissimilarity fig s3analysis of community diversity faith™s phylogenetic diversity index revealed astable and rich microbial community during the naive phase preceding a sharpdecrease following antibiotic treatment and a further decrease immediately followingvrefm colonization fig 2b of note is the responsiveness of the microbiota within h to the removal of antibiotics at the end of day community richness began torebound at approximately days after vrefm colonization ie day with group ademonstrating a higher rate of rebound compared to groups b and c calculating thedistances of dissimilarity unweighted unifrac distances of each mouse microbiotatime point relative to day a proxy for the naive bacterial community phenotyperevealed a small dissimilarity distance for samples collected during the naive phase andan increasing dissimilarity distance following antibiotic treatment day and vrefmcolonization day fig 2c there was a downward trajectory in distance scores daysafter vrefm colonization ie day group a followed a sharper return to a microbiotaresembling day these observations suggest that mice were transitioning toward apersistent carrierlike state and that the rebounding community richness toward levelsrepresentative of the naive phase was by a microbial community structure that resembled the naive phase additional studies where the time frame of postvrefm challengeextends beyond week of monitoring are needed to understand whether the perturbed microbiome will return to resemble an absolute naive state or arrive at a newaltered statemultinomial regression identifies sotus most positively associated with vrefmcolonization multinomial regression using songbird was employed to identify sotusjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig diversity analyses a principalcoordinate analysis plot of unweighted unifrac distances data points areprojected onto the sample space and colored by prevrefm colonization red and postvrefm colonization bluenote that circles and ellipses function to highlight the separation of experimental phases and do not indicatestatistical confidence intervals principal coordinate axis explains of the variation observed between thenaive microbiota and those from the postvrefm colonization phase b community richness of the murine gutmicrobiome as measured by faith™s phylogenetic diversity in response to ceftriaxone treatment and challengewith vrefm c community dissimilarity distances as calculated by unweighted unifrac of each time point relativeto day naive phasethat were most positively and negatively associated with the postvrefm colonizationphase fig the five most positively associated sotus were enterococcus bacteroideserysipelotrichaceae catabacter and lachnospiraceae while the five most negativelyassociated were clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridiales temporal tracking of exact sequence variants esvs demonstrated that theesv feature classified as enterococcus”and identified as the most positively associatedwith the postvrefm colonization phase”was most abundant on days of challengeconfirming that this esv likely was the st796 vrefm colonization challenge anismsfig there were a further eight esv features classified as enterococcus however theywere absent during the days representing vrefm colonization and lacked positiveassociations with the postvrefm colonization phase suggesting that these featuresrepresent murine gut commensal enterococcijulyaugust volume issue e0045220msystemsasm 0cmu fig multinomial regression multinomial regression identified an enterococcus exact sequence variant as the most positivelyassociated with the colonization phase log fold change score of read counts for the enterococcus esv tracked daily acrossthe experiment showing high abundance during the days of vrefm challengemolecular networking identifies differential metabolome profiles duplicatefecal samples from key time points throughout the experiment ie days and were analyzed by datadependent tandem mass spectrometry msms performed on a liquid chromatography quadrupole time of flight lcqtof system tomonitor changes in the murine gut metabolome table polar metabolite analysiswas given preference in an effort to broadly capture primary metabolites that play a keyrole in œmetabolic handoffs that define interspecies interactions analysis of the globalmetabolome profile of each sample was measured based on their overlapping molecules and a pcoa plot using a binary jaccard distance metric through the global naturalproducts social molecular networking gnps platform a separation of metaboliteprofiles along pcoa1 was observed fig 4a metabolomes from the naive andlate vrefm colonization phase tended to cluster together while samples from thepostantibiotic phases including the early vrefm colonization phase clustered togethersupporting pairwise permutational multivariate analysis of variance permanovatesting fig s4 highlights that naive and early vre samples are significantly differentwhile late vre has a lower distance to naive samples compared to antibiotictreatedantibiotic wean and early vre samplesrandom forest analysis of spectrum profiles from lcmsms was used to predictexperimental phase and rank the importance of metabolite association with eachexperimental phase the top metabolite features for each experimental phase arehighlighted in fig 4b unique profiles of metabolite features were observed for eachphase of the experiment importantly the late vrefm colonization phase capturesan unknown metabolite feature with a masstocharge ratio mz of and retention time rt of this metabolite is exclusively present during whatrepresents the transition toward resembling the naive microbiome manual curationoffeature in positiveion mode predicts a molecular formula c5h8n4o3with ±–10 ppm in mass error the major peaks in the msms spectrum for feature are precursor ion [m«¹h]«¹ assumed precursor ion [h2o] product ion [likely c2h2o] [c2h2o«¹h] plus product ionfurther supporting neutral loss of c2h2o given the summation of results the chemicalstructure of feature is likely to contain a nacetylated hydroxyl group peakquantification values indicate its presence during the late phase of vre colonizationfig 4c further manual curation of msms data identified ceftriaxone as feature julyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig metabolomic analyses a emperor plot displaying principalcoordinate analysis of binary jaccard distances of metabolomic profiles samples are colorcoded and the colors represent the naive orange antibiotic treatment red antibiotic weaning blue early vre colonization green and late vrefmcolonization purple phases b random forest classifier identifying metabolite features spectra for each phase of the experiment the heatmap is color codedfrom low ranking score white ie lowest importance to high ranking score dark blue highest importance metabolite features are labeled by theirmasscharge ratios and retention times for the reason that current databases do not capture their chemical structure andor identifications abx tx antibiotictreatment c peak quantification values for feature mz «½ and rt «½ present in abundance during vre colonization late phase dpeak quantification values for ceftriaxone mz «½ and rt «½ tracked across the experiment ceftriaxone values are highest during antibiotictreatment phase and begins to wane during antibiotic weaning phase julyaugust volume issue e0045220msystemsasm 0cmu with an mz of and rt of and mostly abundant during days of antibioticexposure fig 4dbacteroidalesassociated metabolites implicated in latephase postvrefm colonization a distinct profile shift in microbe and metabolite abundances as calculatedby multinomial regression was observed particularly during latephase vrefm colonization fig s6 shallow neural networking analysis with mmvec was used to predictmicrobemetabolite interactions through their cooccurrence probabilities fig sequential biplots captured the shift in experimental phases and highlighted the cooccurrences of microbiota and metabolomic data sets fig 5a to c there was a strongenterococcus effect as indicated by the magnitude of the corresponding arrow and therebounding species during the latephase vrefm colonization are predominantly bacteroidales sotus fig 5c with cooccurring metabolite features mz rt and mz rt metabolite feature mz rt was ranked asbeing highly associated with the postvre colonization phase these results integratemicrobial and metabolite data sets to reveal which microbes may be responsible fordetected metabolites in this instance the metabolite present during the phase representing a transition toward a microbiome approximating the naive state feature mz and rt fig 4b is linked with bacteroidales fig 5adiscussionin this study of the murine gut ecosystem we employed a mouse model ofgastrointestinal tract colonization that replicates the shift in bacterial compositionwhen patients enter the health care system develop an imbalance in their microbiomeas a result of pretreatment eg antibiotic treatment and are subsequently colonizedwith a hospital superbug the resolution of current studies describes a consortiumof commensal microbes that can for example reduce the magnitude of vrefm colonization however understanding the key metabolic shifts relative to the gutmicrobiota remains challenging here we employed amplicon 16s rrna genesequencing and highresolution mass spectrometry metabolomics in an effort towarddetermining microbiotametabolome interactions during vrefm colonization we demonstrated clear changes in the gut microbiome in response to ceftriaxone and vrefmchallengeconceptual and statistical advances in analysis of amplicon 16s rrna gene data whereby otus are clustered at a nucleotide similarity threshold allows for theidentification of exact sequence variants esvs query against an errorcorrecteddatabase can detect multiple esvs that may be classified to the same taxonomicrank for example our analyses identified multiple esvs classified as enterococcihowever when the relative abundances were tracked across the chronology of theexperiment only one enterococcus esv was dominant in relative abundances and mostpositively associated with the days of postvrefm challenge fig this highlights theresolving power to differentiate between commensal and pathogenic strains of enterococci when the composition of the microbial community is considered the factthat this was achievable at the level of amplicon 16s rrna gene sequencing alludes tothe possibility of implementing microbiota screenings as routine diagnostics for patients entering health care systems further firstorder level shifts in microbial community composition was observed in response to ceftriaxone and subsequent vrefmchallenge fig three days after vrefm colonization ie day the microbiomerichness begins to rebound suggesting that mice are transitioning toward a persistentcarrierlike state interestingly the group a cohort exhibited a higher rate of reboundthat may be facilitated by their initially higher microbial community richness andpredominance of lactobacillales on the day of vrefm challenge fig 2b this observation supports the need to prescreen œbaseline microbiota profiles of patients uponadmission into hospital for the reason that it is not necessarily which microbialpopulations are removed postperturbation eg antibiotic pretreatment but insteadwhich populations persist that drives the responding phenotype we can begin toassess patients from across different wards eg intensive care unit oncology neuroljulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vrefig microbemetabolite vector biplots sequential biplots highlighting the changing metabolitedifferentials across each key phase of the experiment abx tx is the antibiotic treatment phase and abxwean is the period when antibiotics were removed for a 24h period prior to colonization with vrefmeach point on the sample space represents metabolites and arrows represent microbes microbe andcontinued on next pagejulyaugust volume issue e0045220msystemsasm 0cmu ogy and healthy cohorts and build a database of microbiome profiles that can be usedas biomarkers to predict i the susceptibility of patients to develop persistent bacterialcolonization and ii propensity to clear the pathogen once colonized the clinicalimplication is that new patients are screened and identified via betadiversity metaanalyses by these biomarkers and placed in bedding cohorts accordingly therebyimproving infectious disease management and isolation precautions within healthcareassociated ecosystemsthe shortlist of microbes ranked as most negatively associated with the colonizationphase clostridiales adlercreutzia mollicutes peptostreptococcaceae and clostridialesfig are hypothesized to play a role in maintaining the health of the animals indeedamong the microbes identified are known shortchain fatty acid eg butyric acidproducers which supports and expands upon those previously identified bycaballero further the use of deblur to identify esvs facilitates the temporaltracking of their relative abundances to inform selection of primary fecal samples thatwill provide the best probability ie highest relative abundance of culturing targettaxa for downstream screening of probiotic potential however translating animalderived observations from experimental animal models to human clinical situationsremains challenging particularly where the key microbes are rodentspecific microbesone solution may be to integrate metabolomics to reveal shared metabolic capacityamong taxonomically divergent microbes our supervised classifying approaches suggests an altered metabolome composition during the late phase of vrefm challengethat may facilitate the apparent œsuppression of vrefm to levels below the limit ofdetection by culture despite the caveat of poor resolution in current databases to linkmetabolite features to associated chemical structures microbemetabolite vector analysis linked metabolite feature mz «½ and rt «½ to bacteroidesfig our efforts toward manually identifying feature suggests a chemicalformula of c5h8n4o3 and a structure likely to contain a nacetylated hydroxyl group aputative annotation through pubchem search is 3hydroxy4nitrosocyanamido butyramide butyramide is the amide group of butyric acid a shortchain fatty acid thathas been shown to play a key role in colonization resistance against intestinal pathogens “ further research to comprehensively characterize interactions betweenmicrobe and metabolites will be critical to address the gaps in our understanding of thebiochemical parameters that define interspecies microbiome interactions during antibiotic pretreatment and persistent infectionsthe resolution of our results provides the basis in which to begin to identifynonantibiotic alternatives to engineer the gut microbiome through prebiotic interventions eg butyric acid and translating animal studies to humanrelevant therapeuticapplications by delineating taxonomically diverse microbes with shared metaboliccapacity here achieving integrative omics to link microbemetabolite associations ourfindings add support to the incorporation of microbiome profiling approaches intoroutine clinical microbiology particularly in the context of monitoring the impacts ofantibiotic usematerials and methodsmouse gastrointestinal colonization model sixweekold wildtype c57bl6 male mice were usedto establish an animal model of gastrointestinal colonization with vrefm mice were cohoused and hadfree access to food ordinary chow and water and had environmental enrichment eg fun tunnels chewblocks and tissue paper the lightdark cycle was 12h light12h dark and cages were changed weeklyfig legend continuedmetabolite features are fixed upon the sample space with gradient coloring of metabolites indicating thetransition across key phases of the experiment the distance between each point is indicative ofmetabolite cooccurrence frequency and the angle between arrows indicates microbial cooccurrencethe directionality of the arrows describes the variation in the metabolites explained by the microbesrepresented by the arrows for example metabolite feature mz and rt isdemonstrated to cooccur with bacteroides information about the abundances of these cooccurringfeatures are provided as heatmaps in fig s6 in the supplemental materialjulyaugust volume issue e0045220msystemsasm 0cgut microbiome and colonization with vremice were pretreated with gliter ceftriaxone in drinking water for days followed by an antibioticwean period of h mice were then challenged with «» cfu vrefm st796genomic dna extraction and sequencing wholecommunity genomic dna gdna was extractedfrom mouse fecal samples using the qiagen powersoil dna extraction kit formerly mobio following themanufacturer™s protocol a preprocessing step of mechanicallysis was incorporated using a bertintechnologies precellysis machine for one round of a 40s cycle at rpm the v4 region of thebacterial 16s rrna gene was amplified using small subunit forward golaybarcoded and ssu806reverse primers following the earth microbiome project protocol and sequenced using the illuminamiseq platform v2 cycles illumina inc san diego ca usa further primary derived data egbiom tables used to produce results can be found within qiita study id amplicon 16s rrna gene profiling analyses sequence data were processed within the qiitav010 framework for quality control split libraries v q2191 demultiplexing trimming sequencereads to a length of nucleotides nt and picking suboperational taxonomic units sotus usingdeblur v110 to resolve singlenucleotide community sequencing patterns ie feature identification ofsotus [] the output biom files were further processed using qiime2 v20197 for downstreamstatistical analyses alpha rarefaction curves were generated to determine whether each sample hadbeen sequenced to saturation the feature table was subsequently rarefied to reads per sampletaxonomy was assigned using the sklearn classifier and greengenes otus from 515f806rregion of sequences classifier available from docsqiime220184dataresources furthermorerelative abundances of each taxa were visualized as bar plots using the qiime2 taxa plugin a phylogenetic tree was constructed using fragment insertion qiime fragmentinsertion sepp [] to guidephylogeneticaware statistical analyses generated using the qiime2 plugin q2diversity coremetricsphylogenetic key metrics computed by default include both alphadiversity eg shannon™s diversityindex faith™s phylogenetic diversity and evenness and betadiversity eg braycurtis distance andunweighted unifrac distance metrics the unweighted unifrac distance matrix was used tocompute first distances and calculate distances relative to day as the baseline between sequentialstates qiime longitudinal firstdistances ggplot2 r v360 ggplot2tidyverse was used tovisualize the distance scores as line plots emperor was used to visualize principalcoordinate analysisplots of unweighted unifrac distances permutationbased statistical testing permanova on unweighted unifrac distances was used to determine whether samples grouped by phase of experimentwere significantly different from one another q2betagroupsignificance songbird githubcommortonjtsongbird was employed to determine the importance ie fold change of each sotu inrelation to a given metadata variable eg vrefm colonization microbial features from all samples weresplit into training and test sets for supervised learning classifier analyses of input samples wereallocated to train the random forest classifier within qiime2 the estimator method used for sampleprediction the different experimental phases were the response variables while the 16s rrna gene datawere the featuresmetabolite extraction and liquid chromatographytandem mass spectrometry analysis duplicate fecal samples as outlined in table were processed for polar metabolite extraction and analysisdays and feces were metabolically arrested by immediate collection into dry ice andstored at “ °c until further processing metabolite extraction from the fecal samples was undertaken bythe addition of \u242el per sample of methanolwater solution [volvol] containing \u242em [13c]sorbitoland \u242em [13c15n]valine and \u242em [13c]leucine as internal standards fecal samples were homogenizedat rpm for min in a thermomixer maintained at °c mechanically disrupted and incubated fora further min in the thermomixer samples were randomized for metabolite extractionmetabolite analysis of the extracted samples pooled biological quality control pbqc samples and mixtures of authentic standard mixes was performed by liquid chromatographymass spectrometrylcms using hydrophilic interaction column zicphilic and highresolution agilent seriesquadrupole time of flight mass spectrometry qtof ms as described previously pcoa of binaryjaccard distances of test standard mixes and pbqc samples are presented in fig s5 in the supplementalmaterial ions were analyzed in positive mode with full scan range of to mz and in datadependent tandem ms mode to facilitate downstream metabolite identificationmetabolomic analyses
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Cabozantinib is an oral multikinase inhibitor whose targets include vascular endothelial growth factor receptors MET and the TAM family of kinases TYRO3 AXL MER Cabozantinib is approved for patients with advanced hepatocellular carcinoma who have been previously treated with sorafenib based on improved overall survival and progressionfree survival relative to placebo in the phase III CELESTIAL study During CELESTIAL the most common adverse events AEs experienced by patients receiving cabozantinib included palmarplantar erythrodysesthesia fatigue gastrointestinalrelated events and hypertension These AEs can significantly impact treatment tolerability and patient quality of life However AEs can be effectively managed with supportive care and dose modifications During CELESTIAL more than half of the patients receiving cabozantinib required a dose reduction while the rate of treatment discontinuation due to AEs was low Here we review the safety profile of cabozantinib and provide guidance on the prevention and management of the more common AEs based on current evidence from the literature as well as our clinical experience We consider the specific challenges faced by clinicians in treating this patient population and discuss factors that may affect exposure and tolerability to cabozantinib IntroductionThere has been a marked increase in liver cancer deaths in recent years In there were a0 new cases of liver cancer worldwide and liver cancer accounted for almost deaths making it the sixth most prevalent cancer worldwide [] The most common primary malignancy of the liver is hepatocellular carcinoma HCC [] The frequency burden and etiology of HCC vary across geographic regions and populations but are linked to prevalence of predisposing chronic hepatic conditions such as Electronic supplementary material The online version of this s1152 contains supplementary material which is available to authorized usersKey Points Cabozantinib represents a treatment option for patients with advanced hepatocellular carcinoma who progress after sorafenibAdverse events associated with cabozantinib may be effectively managed with supportive care and dose modifications thereby allowing patients to continue treatment at the appropriate dose with minimum interruptionStudies of cabozantinib in the firstline setting are ongoing by understanding the safety profile of this drug clinicians will be able to balance efficacy with tolerability for each patient Gabriel Schwartz GabrielSchwartzucsfedu Gastrointestinal Medical Oncology Clinic University of a0California San Francisco Fourth St Fourth Floor San a0Francisco CA a0 USAIndiana University Health Simon Cancer Center Indianapolis IN USA Department of a0Medicine University of a0California San Francisco San a0Francisco CA USAIRCCS Istituto Clinico Humanitas Rozzano Milan Italy viral hepatitis and nonalcoholic fatty liver disease NAFLD or nonalcoholic steatohepatitis NASH which generally develop in the setting of cirrhosis [ ] In recent years the incidence of nonviral HCC has increased while the proportion of HCC cases related to viral hepatitis has declined [ ] Additional risk factors for HCC include alcohol consumption smoking obesity and diabetes [] As the epidemiology of these conditions has evolved so too has the etiology of HCC []Vol0123456789 0c G a0Schwartz et alFor patients with advanced HCC the vascular endothelial growth factor receptor VEGFR“targeting tyrosine kinase inhibitor TKI sorafenib has been a standard of care [] however the treatment landscape has been transformed in recent years with the introduction of newer TKIs immunotherapies and monoclonal antibody therapies [] This provides clinicians and patients with a variety of treatment options based on mechanism of action and safety profileCabozantinib is a multikinase inhibitor that targets VEGFR “ MET the TAM family of kinases TYRO3 AXL MER RET ROS1 KIT TRKB FLT3 and TIE2 [ ] several of which are implicated in tumor growth angiogenesis and immune regulation [] VEGFR MET and AXL have been implicated in the pathogenesis of HCC [“] A capsule formulation of cabozantinib was first approved in for treatment of progressive metastatic medullary thyroid carcinoma MTC [] The tablet formulation not bioequivalent or interchangeable with the capsule [] was subsequently approved for patients with advanced renal cell carcinoma RCC [ ] and more recently for patients with advanced HCC who have received prior sorafenib [ ] The approval in HCC was based on outcomes from the pivotal phase III CELESTIAL trial which showed significantly improved overall survival OS and progressionfree survival PFS with cabozantinib relative to placebo in patients who received prior sorafenib [] The safety profile of cabozantinib was manageable nearly all patients receiving cabozantinib experienced an adverse event AE but these were effectively managed with dose modification and supportive care measuresClinicians treating patients with advanced HCC can face significant challenges as many patients present with cirrhosis and comorbidities that can impact treatment tolerability Adequate assessment of liver function and management of comorbidities are therefore essential before and during HCC treatment [] Here we provide guidance on the management of AEs associated with cabozantinib in patients with advanced HCC We briefly review outcomes from CELESTIAL and focus on managing some of the more common AEs experienced by patients based on current evidence from the literature as well as our own clinical experience Cabozantinib in a0Hepatocellular Carcinoma CELESTIALIn the phase III CELESTIAL study patients with advanced HCC were randomized to treatment with cabozantinib a0mg daily or placebo [] Patients were required to have had prior treatment with sorafenib and could have received up to two prior systemic regimens for HCC Eastern Cooperative Oncology Group ECOG performance status PS of or and ChildPugh class A liver function see Electronic Supplementary Table a0 for definition were also required At the second planned interim analysis patients had been randomized The study met its primary endpoint with significantly improved OS with cabozantinib relative to placebo median OS was versus months hazard ratio confidence interval [CI] “ p a0 a0 Cabozantinib also improved PFS with a median of versus months hazard ratio CI “ p a0 a0 as well the objective response rate per Response Evaluation Criteria In Solid Tumors RECIST v11 vs a0 p a0 a0 Safety and a0TolerabilityAllcause AE rates were generally higher in the cabozantinib arm than in the placebo arm some of the more common AEs experienced by patients in the cabozantinib a0 versus placebo arms included diarrhea vs decreased appetite vs palmarplantar erythrodysesthesia PPE vs fatigue vs nausea vs hypertension vs vomiting vs asthenia vs and increased aspartate aminotransferase AST vs Fig a0 The most common grade AEs in the cabozantinib versus placebo arms were PPE vs hypertension vs increased AST vs fatigue vs and diarrhea vs Overall the safety profile of cabozantinib was consistent with those from the phase III studies in RCC and MTC with gastrointestinal GI events PPE fatigue and hypertension being the most common AEs experienced by patients across studies [ ]In addition to supportive care measures protocolspecified dose modification including dose interruption and reduction was utilized to manage AEs [] Eightyfour percent of patients in the cabozantinib arm had an AE that led to dose interruption and had a dose interruption due to a grade AE [] Sixtytwo percent of patients had at least one dose reduction due to an AE [] and dose reduced due to a grade AE [] Thirtythree percent of patients had a second dose reduction [] Median time to first and second dose reduction in the cabozantinib arm was a0days and a0days respectively PPE was the event that most commonly led to dose interruption and dose reduction followed by diarrhea and and fatigue and [] Although most patients receiving cabozantinib required a dose interruption the rate of discontinuation due to treatmentrelated AEs was relatively low in the cabozantinib arm vs in the placebo arm indicating that the majority of AEs were adequately managed with dose modification and supportive care In the cabozantinib group AEs that led to treatment discontinuation in ‰¥ a0 of patients were PPE fatigue decreased appetite diarrhea and nausea In a subgroup analysis of patients 0cAE Any grade [Grade Grade ]Fatigue [ ]Hypertension [ ]Increased AST [ ]Increased ALT [ ]Asthenia [ ]Nausea [ ]Vomiting [ ]Decrease appetite [ ]Weight loss [ ]Diarrhea [ ]Constipation [ ]Abdominal pain [ ]PPE [ ]Fig Incidence rates for select AEs experienced by patients with HCC receiving cabozantinib during the CELESTIAL trial [] AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders AE adverse event ALT alanine aminotransferase AST aspartate aminotransferase HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesiawho received sorafenib as the only prior treatment for HCC duration of prior sorafenib did not appear to impact the types or rates of grade AEs []Generally the more common AEs emerged in the first weeks of treatment Fig a0 However clinicians should be aware of infrequent or serious events that can occur in the later phases of treatment Hemorrhagic events of grade or higher were reported in of patients in the cabozantinib arm including five patients with a grade event Bleeding complications are associated with antiangiogenic therapies and may arise as a result of reduced vascular integrity [] Median time to onset of hemorrhagic events was a0weeks in CELESTIAL Other grade or higher rare but serious AEs in patients receiving cabozantinib included fistulas of patients GI perforations and arterial and venous or mixed thrombotic events [] Median time to first occurrence was approximately a0weeks for GI perforations weeks for venous and arterial thromboembolisms and weeks for fistulas [] Two patients in the cabozantinib arm had developed ChildPugh C ie decompensated cirrhosis by the week assessment []Reversible posterior leukoencephalopathy syndrome RPLS a syndrome of subcortical vasogenic edema diagnosed by magnetic resonance imaging has been reported with cabozantinib and other TKIs [ ] Although there were no RPLS events during CELESTIAL [] clinicians should be aware of the symptoms which include headaches seizures confusion changes to vision or altered mental function [ ] Osteonecrosis of the jaw ONJ whereby necrotic jaw bone becomes exposed is another rare but serious AE associated with TKIs including cabozantinib [“] although again there were no ONJ events reported during this study [] The use of antiresorptive drugs in patients with bone metastases is also associated with development of ONJ []A post hoc analysis estimated the incremental qualityadjusted lifeyears accrued with cabozantinib compared with placebo using the fivedimension fivelevel EuroQol questionnaire [] Cabozantinib treatment was associated with an initial decline in mean total qualityadjusted lifeyears during the first “ a0months relative to placebo followed by longterm improvement that was significantly greater than that observed with placebo p a0 a0Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c Fig Rates and timing of select AEs in patients with HCC receiving cabozantinib during the CELESTIAL trial The size of the circle is proportional to the AE rate AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders black generalother AE adverse event ATE arterial thrombotic event GI gastrointestinal GR grade HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesia VTE venous thrombotic eventMedian time to first dosereduction to mg weeksG a0Schwartz et alMedian time to seconddose reduction to mg weeksFistulas Hemorrhage GR ATEs VTE Wound complication GI perforations Hepatic encephalopathy Diarrhea PPE Hypertension Median time to first occurrence weeks Factors Affecting Tolerability of a0Cabozantinib Co‘morbiditiesHCC emerges primarily in older adults [] In addition to the underlying HCC etiology older adults with HCC are likely to have additional comorbidities such as cardiovascular or pulmonary disease [] and it is not uncommon for patients with HCC to have multiple comorbidities [] Liver cirrhosis with compromised liver function and decreased hepatic reserve is a major risk factor for HCC development Other HCCrelated comorbidities include hepatitis B virushepatitis C virus infection alcoholic liver disease NASH and diabetes [] In addition metabolic syndrome characterized by hyperlipidemia and hypertension is linked to development of NAFLD which may progress to NASH cirrhosis and finally HCC [] For patients with HCC assessment of liver function is a key step in treatment decisionmaking [] Patients with moderate or severe hepatic impairment are predominantly excluded from clinical trials in HCC therefore treatment of these patients is complicated by a lack of prospective clinical data as well as competing comorbidities []Although the number of patients with HCC and prior an transplant is limited these patients are generally excluded from clinical trials and treatment is complicated by the need for immunosuppression TKIs may be used to treat posttransplant HCC recurrence although supporting data are limited The use of TKIs in these patients is complex so treatment decisions should involve collaboration between the oncology and transplant medicine care teams The use of sorafenib in patients receiving mammalian target of rapamycin inhibitorbased immunosuppression has been associated with an increased risk of fatal bleeding [ ] Immunotherapies are associated with an increased risk of an rejection in posttransplant patients [] Cabozantinib Clearance and a0ExposureTKIs are associated with high interpatient variability in clearance and exposure which may affect both efficacy and tolerability This variability may be due to a variety of factors including genetic background drug“drug interactions drugfood interactions and renal or hepatic impairment [] As evidenced by exposureresponse modeling patients with low clearance of cabozantinib may have higher exposure and an increased risk of developing certain AEs [ ] Awareness of these nuances may help clinicians to mitigate their effects thereby balancing efficacy with tolerability Hepatic and a0Renal ImpairmentAccording to pharmacokinetic analyses of patients with HCC and other tumor types mild hepatic impairment is predicted to have a minimal effect on cabozantinib exposure [] therefore adjustment of the recommended 60mg starting dose is not necessary for patients with Child“Pugh A 0cliver function [ ] Data on the pharmacokinetics of cabozantinib in patients with moderate ChildPugh B or severe Child“Pugh C hepatic impairment are limited [] As per the US Food and Drug Administration FDA prescribing information the starting dose of cabozantinib should be reduced to mg in patients with moderate hepatic impairment while cabozantinib is not recommended for patients with severe hepatic impairment [] Note that the European Summary of Product Characteristics SmPC does not recommend dose adjustments for moderate hepatic impairment owing to limited data [] For patients with HCC increased exposure due to hepatic impairment should be considered if intolerable AEs develop and dose modification undertaken as recommended Fig a0 [ ] Cabozantinib should be used with caution in patients with mild or moderate renal impairment owing to the potential for increased exposure although no dose adjustments are necessary Cabozantinib is not recommended for use in patients with severe renal impairment owing to lack of data on safety and efficacy in this population [ ] Drug“Drug and a0Drug“Food InteractionsGiven the range of comorbidities that may exist in patients with advanced HCC it is important to review all concomitant medications for potential interactions prior to initiation of treatment with cabozantinib Certain medications and foods have been shown to modulate the pharmacokinetics of cabozantinib which may in turn impact exposure levels efficacy and risk of AEs Cabozantinib is metabolized in the liver primarily by the enzyme cytochrome P450 3A4 CYP3A4 [] therefore CYP3A4 inhibitors or inducers may impact exposure examples of CYP3A4 inducersinhibitors are shown in Electronic Supplementary Table a0 Strong CYP3A4 inhibitorsinducers should be avoided in patients receiving cabozantinib If concomitant administration of a strong CYP3A4 inhibitor is necessary then the cabozantinib Recommended dose at initiation mg Except for¢ Patients with moderate hepatic impairment or coadministration of a strong CYP3A4 inhibitor initiate cabozantinib at mg ¢ Patients with coadministration of a strong CYP3A4 inducer initiate cabozantinib at mg Safety assessmentNo AEsGrade Grade AE or ONJSupportive caresee Tables “DosemodificationImprovementtolerableelbarelotnIlitnu esod dloHgrade ‰¤Continue at tolerated doseReduce dose by mg and restart mg †’ mg mg †’ mg mg †’ mg mg †’ mg or discontinueImmediate Discontinuation¢ Severe hemorrhage¢ Development of GI perforation or unmanageable fistula¢ Serious thromboembolic event eg myocardial infarction cerebral infarction¢ Hypertensive crisis or severe hypertension despite optimal medical management¢ Nephrotic syndrome¢ Reversible posterior leukoencephalopathy syndromeFig Cabozantinib dosing algorithm [ ] AE adverse event CYP3A4 cytochrome P450 3A4 GI gastrointestinal ONJ osteonecrosis of the jawManagement of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et aldose should be reduced by a0mg for example from to a0mg [] Conversely the cabozantinib dose should be increased by a0mg if strong CYP3A4 inducers need to be coadministered [ ]Cabozantinib should not be taken with any food as this may affect absorption [] The label recommends that cabozantinib be taken at least h before or at least h after eating [] Grapefruit and grapefruit juice are strong CYP3A4 inhibitors and should be avoided [ ]Cabozantinib may be used with caution in patients who are receiving concurrent antiarrhythmics or other QTprolonging agents [] This is based on a study of patients with MTC who received a daily 140mg capsule dose of cabozantinib recommended for this indication in which the mean deltadelta QT interval was increased by approximately “ a0ms with upper CIs not exceeding ms [] Such an increase is within the range considered to be acceptable for oncology drugs in this setting [] No patient in the aforementioned study or in CELESTIAL had a confirmed QTcF QT corrected using Fridericia™s method a0 ms [] which is considered clinically significant [] For patients receiving cabozantinib monitoring with periodic electrocardiogram and electrolyte measurements may be advisable particularly in patients with risk factors such as cardiac disease or a prior history of QT prolongation [] Concomitant use of proton pump inhibitors PPIs such as esomeprazole does not affect cabozantinib exposure levels [] However PPIs may cause hypomagnesemia which is linked to an increased risk of QT prolongation [] Therefore coadministration of PPIs and cabozantinib should be undertaken with caution following an individualized assessment of the patient™s baseline magnesium levels and concomitant medications that may also influence QT Pretreatment AssessmentsGiven the heterogeneity of the HCC patient population and the complexity associated with comorbidities and concomitant medications all patients should undergo a comprehensive assessment of medical history prior to initiation of treatment with cabozantinib Ideally the multidisciplinary care team should include an oncology pharmacist [] A œbrown bag medication review should be carried out prior to treatment initiation [] whereby the patient brings in all current medications including overthecounter medicines vitamins herbal remedies etc Therapeutic duplications should be eliminated for example concomitant PPIs and histamine H2 antagonists H2 blockers Switching and deprescribing should be considered where possible to minimize the risk of drugdrug interactions Adverse Event ManagementThe AE profile of cabozantinib is generally similar to that of other VEGFRtargeting TKIs with GIrelated AEs fatigue PPE and hypertension being the most common AEs [] Other AEs that occur less frequently can also have a significant impact on quality of life QoL and treatment adherence such as mucosal inflammation [] Hepatobiliary AEs such as elevated AST alanine aminotransferase ALT and bilirubin are particularly relevant in the context of advanced HCC and need to be carefully monitoredProphylactic and supportive care measures for the more common cabozantinibassociated AEs grade or tolerable grade are outlined in Tables a0 and discussed in the upcoming sections Symptom gradings are summarized in Electronic Supplementary Table a0 Dose interruption is recommended for management of intolerable grade AEs not resolved with supportive care measures or for any grade AEs Fig a0 [ ] Cabozantinib may be reinitiated at a reduced dose once the event resolves to grade ‰¤ a0 Palmar“Plantar ErythrodysesthesiaPPE is one of the more common events associated with anticancer therapies including VEGFRtargeting multikinase inhibitors [“] PPE is characterized by pain redness tingling and swelling of hands and feet [] Presentation may vary according to the etiologic agent PPE induced by TKIs is typically localized to pressurebearing areas in contrast to that caused by chemotherapy which has a more diffuse pattern It has been hypothesized that inhibition of multiple angiogenic pathways by TKIs may compromise repair of capillary microtrauma in areas exposed to mechanical stress such as the hands and feet [ ] Although not lifethreatening PPE can rapidly progress to a debilitating condition negatively impacting QoL [ ]Prophylaxis and prompt management of emerging symptoms may help to minimize the impact of PPE on QoL and adherence Table a0 Prophylactic measures predominantly involve skin care practices to remove hyperkeratotic areas and to minimize friction and damage prior to the start of treatment [ ] Recommendations include use of thick cotton gloves and socks padded insoles in shoes and avoidance of heat or friction on the hands and feet [ ] Patients with potentially predisposing comorbidities such as peripheral neuropathy [ ] as well as patients with persistent symptoms may benefit from involvement of a podiatrist andor dermatologist within their multidisciplinary care team [] Treatment strategies involve moisturization prevention of infection and analgesia [ ] Monitoring is crucial so that emerging symptoms can be proactively managed Patients should be assessed at baseline 0cTable Adverse event management strategies”palmar“plantar erythrodysesthesia PPE PPEProphylaxisProvide education on prophylactic skin care before starting treatment []Advise manicure and pedicure before and during treatment to remove hyperkeratotic areas [ ]Protect sensitive areas recommend sunscreen with SPF protection ‰¥ a0 thick cotton gloves and socks padded insoles and wellfitting shoes avoid heat sources and use cooling aids and avoid activities that may cause force or rubbing on the hands and feet eg heavy lifting dish washing [ ] delegate such tasks to caregiversAdvise on optimal hand cleaning avoid fragrancedfoaming soaps and hand sanitizers containing alcohol ensure hands are dried thoroughly after cleaning []Prophylactically administer keratolytic cream eg urea [ ]Monitor regularly in order to proactively manage skin toxicities evaluate at baseline monitor up to weekly for the first “ months and monthly thereafter [ ]Supportive careContinue prophylactic measures []Maintain moisture of skin using emollients [ ]Consider topical treatment with salicylic acid urea “ cream either alone or with tazarotene cream or fluorouracil cream andor clobetasol cream topical analgesics may be added for pain control [ ]Topical cortisone and clobetasol may also be used consider oral analgesics eg NSAIDs pregabalin cautious use of opioids [ ]Consult with a dermatologist to drain blisters and remove hyperkeratotic areas []To prevent infection of cracked skin soak in equal parts vinegar and water for min per day [] a0Antibiotics should be prescribed only if there is evidence of infection [] a0There is limited evidence for the use of pyridoxine vitamin B6 []NSAID nonsteroidal antiinflammatory drug SPF sun protection factorTable Adverse event management strategies”fatigue FatigueProphylaxisProvide patient education about fatigue management tools and available support []Establish baseline fatigue levels with a fatigue scale and remeasure regularly during patient visits []Ensure adequate fluid and nutritional intake []Advise behavioral modifications balancing rest with physical activity recommendations include relaxation massage yoga aerobic or resistance exercise programs and energy conservation strategies [“]Assess thyroid function prior to treatment and monitor during treatment [ ]Supportive careRule out alternative causes of fatigue eg anemia endocrine disorders such as hypothyroidism pain dehydration hypercalcemia or depressionanxiety [ ]Advise patient to increase activity consider referral to a physical therapist []Consider referral to nutritional counselor for nutritional therapy []Incorporate psychosocial measures including cognitive therapy social support biofeedback and sleep therapy []Incorporate management with psychostimulants eg methylphenidate [ ] or corticosteroids eg methylprednisolone []Owing to effects on CYP3A45 substrates including cabozantinib longterm use of modafinil should be avoided []CYP3A4 cytochrome P450 3A4 CYP2C19 cytochrome P450 2C19Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et alTable Adverse event management strategies”gastrointestinal GastrointestinalDiarrheaProphylaxisInstruct patients to monitor food and fluid intake [] a0Recommended water intake per day from all beverages and food [] L oz for women L oz for men a0Advise patients to keep a stool diary and to promptly report diarrhea to their healthcare provider [ ]Advise patients to avoid foods that may cause GI events such as lactosecontaining foods caffeine highfat or highfiber food eg nuts seeds legumes and raw fruit and vegetables [ ]Implement dehydration prevention management through oral rehydration with electrolytes []Supportive careAdminister loperamide at the first sign of diarrhea [ ] a0 mg orally followed by mg every h until “ h after last bowel movement maximum of mg in h a0For chronic diarrhea “ mg twice daily titrated as needed a0Alternatives to loperamide include diphenoxylate and tincture of opium []Implement supportive dietary modifications continuous oral hydration correction of fluid and electrolytes small frequent meals avoid lactosecontaining food and drink [ ] a0The BRAT bananas rice applesauce toast diet may help to alleviate mild diarrhea []If there are signs of severe dehydration administer IV fluid replacement isotonic saline or balanced salt solution []Rule out nontreatmentrelated causes eg infectious diarrhea []Decreased appetiteProphylaxisAdvise patients to monitor their appetite and weight []Encourage patients to consume highprotein calorierich food fruit and vegetables nutritional supplements that they may snack on throughout the day [ ]Advise patients to preprepare and freeze nutritional preferred food []Supportive careTreat underlying nausea []Consider involving a dietitian who may recommend scheduled eating times []Recommend a highcalorie diet []Provide dietary education alongside dietary modifications andor nutritionalvitamin supplements []Use a pharmacologic agent to stimulate appetite such as a CB1 receptor agonist dronabinol [ ] systemic corticosteroid methylprednisolone [ ] progestin megestrol acetate [ ] or mirtazapine [ ]NauseavomitingProphylaxisAssess risk factors for nauseavomiting prior to treatment []Metoclopramide may be administered prophylactically []Advise patients to avoid foods that are overly sweet greasy fried or spicy []Supportive careAntiemetic agents such as dopamine receptor antagonists eg metoclopramide prochlorperazine or 5HT3 receptor agonists eg ondansetron are recommended for management of nausea or vomiting [ ] a0Certain NK1 receptor agonists eg aprepitant and netupitant and dexamethasone are inducers inhibitors andor substrates of CYP3A4 and thus could alter cabozantinib exposure [ ] however the potential for ondansetron to prolong the QT interval must also be considered [] There is moderate evidence for olanzapine an antipsychotic drug that blocks multiple neurotransmitters as an antiemetic in this setting [] 0cTable continuedMucosal inflammationstomatitisProphylaxisA comprehensive dental examination should be conducted prior to treatment to identify potential complications []Mitigation of potential risk factors [ ] a0Modification of illfitting dentures a0Appropriate care for preexisting dental problems such as caries ulcers etcRegular oral assessments should be conducted throughout treatment [ ]Educate patients on good oral hygiene and oral care protocols including written instructions [] a0The oral cavity should be washed using salinecontaining mouthwash up to four times daily and dentures should be regularly cleaned []Painful stimuli eg smoking alcohol hot fooddrink sharp or spicy food should be avoided [ ]Supportive careTreat pain with doxepin mouthwash or viscous lidocaine [ ]Lactobacillus lozenges may be used to reduce inflammation []Obtain bacterialviral culture if oral infection is suspected and treat infection as clinically indicated []5HT3 5hydroxytryptamine CB1 cannabinoid CYP3A4 cytochrome P450 3A4 GI gastrointestinal IV intravenous NK neurokininTable Adverse event management strategies”hypertension HypertensionProphylaxisMonitor BP before initiation of cabozantinib using a minimum of two standardized BP measurements alongside patient history physical assessment directed laboratory evaluation and an instrument test to determine cardiovascular risk factors [ ]Educate patients on BP selfmonitoring and advise they keep a BP log []BP should be well controlled prior to initiating cabozantinib ensure patients who have already been prescribed antihypertensive therapy are adherent and that therapy has been titrated to effective doses [ ]Check for potential drugdrug interactions of existing antihypertensive agents with cabozantinib Supplementary Table a0Consider effects of concomitant medications on BP eg antiinflammatory drugs can increase BP opiates can lower BP []Monitor BP during cabozantinib treatment weekly during first cycle every ‰¥ a0“ weeks thereafter []Supportive careAdd antihypertensive medications or increase dose of existing medication as indicated [ ]Patients with portal hypertension should be treated with nonselective betablockers []The antihypertensive agent should be carefully considered owing to potential inhibition of CYP3A4 [ ] Supplementary Table a0 a0Thiazides angiotensinconverting enzyme inhibitors and angiotensin receptor blockers may be used to treat hypertension and are not known CYP3A4 substrates [“ ] a0Thiazide diuretics should be prescribed with caution owing to the associated risk of diarrhea [] a0Diltiazem and verapamil are moderate inhibitors of CYP3A4 [] a0Amlodipine felodipine lercanidipine nisoldipine and nifedipine are not considered to be CYP3A4 inhibitors []BP blood pressure CYP3A4 cytochrome P450 3A4and monitored at least weekly for the first “ months of treatment and monthly thereafter [ ] Close monitoring in the early stages of treatment need not involve weekly visits”phone calls from a clinician nurse or pharmacist may facilitate monitoring in between scheduled appointments [] Patients should be encouraged to report early signs of PPE to their healthcare provider [] it may also be reassuring for patients to know that early reporting and management of AEs
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there is currently a major human pandemic caused by the novelsevere acute respiratory syndrome sars coronavirus2 sarscov2 that leads to coronavirusinduced disease covid191 itis primarily a viralinduced ‚ammatory disease of the airwaysand lungs that causes severe respiratory issues sarscov2 usesthe angiotensin converting enzymeii receptor ace2 to bindand infect cells leading to internalization and proliferation23‚ammatoryinnate and adaptive immune responses areinduced to clear the virus but also cause host tissue damage45consequent hypoxia leads to systemic involvement particularlyofleads to vasoconstriction reducedperfusion and an failure6 much remains to be understoodof the ‚ammatory and immune responses that are induced bythe infection and how they induce pathogenesis ventilation andoxygen therapy are primary treatments and it is emerging thatthose with severe disease who survive develop lung fibrosis7 themost effective pharmacological treatments remain illdefinedwith varying results with hydroxychloroquine8 but more promising results with dexamethasone9the vasculature thatelucidating the mechanisms of pathogenesis will enable theidentification of the most effective therapies animal models ofsarscov2 infection and covid19 that recapitulate the hallmarkfeatures of the human disease will undoubtedly be valuable inelucidating pathogenic mechanisms identifying new therapeutictargets and developing and testing new and effective treatmentshuman infection and diseasesarscov2 is a betacoronavirus closely related to sarscovthat caused a relatively small outbreak in the early 2000s210similar to sarscov sarscov2 binds the ace2 receptor andrequires proteases such as serine tmprss2 to cleave the viralspike s protein required for sarscov and sarscov21112 cellentry2 this step may be facilitated by endosomal proteases suchas cathepsinl and enhanced by the protein furin13 the virusthen enters the host cell by endocytosisa critical element of sarscov2 tropism in humans is theabundance of ace2 in the upper respiratory tract urt especiallythe nasopharynx14 the molecular configuration of the sarscov21centre for ‚ammation centenary institute and university of technology sydney faculty of science sydney australia 2zhejiang universityuniversity of edinburgh institutezhejiang university school of medicine zju international campus haining china 3second affiliated hospital zhejiang university school of medicine hangzhou china4department of genomes and genetics institut pasteur paris france 5centre for innate immunity and infectious diseases hudson institute of medical research clayton vicaustralia 6department of molecular and translational sciences monash university clayton vic australia 7ritchie centre hudson institute of medical research clayton vic australia 8department of paediatrics monash university clayton vic australia 9monash newborn monash children™s hospital clayton vic australia 10priorityresearch centre for healthy lungs hunter medical research institute and university of newcastle newcastle nsw australia 11school of chemistry and molecular biosciencesand australian infectious diseases research centre the university of queensland brisbane australia 12centenary institute and dermatology the university of sydney andcancer services royal prince alfred hospital sydney nsw australia 13centenary institute and faculty of medicine and health university of sydney sydney australia 14dr johnand anne chong lab for functional genomics charles perkins centre centenary institute and school of life and environmental sciences university of sydney sydney nswaustralia 15charles perkins centre and school of life and environmental sciences university of sydney and faculty of medicine and health concord clinical school anzacresearch institute and centre for education and research on ageing sydney australia 16discipline of infectious diseases and immunology central clinical school faculty ofmedicine and health and the charles perkins centre the university of sydney camperdown sydney australia 17woolcock institute of medical research sydney australia and18centenary institute the university of sydney and department of clinical immunology royal prince alfred hospital sydney nsw australiacorrespondence p m hansbro philiphansbroutseduaureceived july revised july accepted july society for mucosal immunology 0canimal and translational models of sarscov2 infection and covid19md johansen membrane binding component of the s protein binds with greateraffinity to ace2 than does sarscov which likely contributes to thehigher infectivity of the former15 the clinical course commenceswith an incubation period with a median of days with illnesstypically developing by days16 this phase is characterized bymild symptoms with most people remaining asymptomatic andinfection thought to be confined to the urt although they arecapable of transmitting infection symptoms when they do occur aretypically acute viral respiratory illness with fever cough dyspnoeafatigue anosmia myalgia and confusion17 in of people thecourse remains mild and disease does not extend to the lowerrespiratory tractlrt however develop more severesymptoms with diffuse widespread pneumonia with havingsevere gas exchange problems acute lung injury and progress ontosyndrome ards1819 the clearestacute respiratory distresspredictor of mortality is age with the case fatality rate risingdramatically over years of age20 other predisposing factors forheightened mortality are male sex social deprivation and chronicdisease particularly chronic obstructive pulmonary disease copdcardiovascular disease cvd obesity and diabetes21a key issue is why some individuals progress to more severe lowerrespiratory disease but others do not one factor is the ability of the‚ammatory and immune responses to confine the infection to theurt ace2 is expressed in the lrt but at lower levels than in thenasopharynx22 also while ciliated airway epithelial cells are readilyinfected and transmit to surrounding cells the reduction in ace2may be a barrier to lrt infectionin those that progress severesystemic ‚ammatory response or œcytokine storm develop thepneumonia associated with severe infection bears all the pathological features of ards with diffuse alveolar damageinterstitialpneumonitis and lymphocytic ltrates2324 unique features ofcritical disease are extravascular fibrin deposition neutrophiltrapping microvascular thrombosis and large vessel pulmonaryemboli24 widespread thrombosis and microangiopathy in criticalcovid19 occurs at higher rates than in ards associated with‚uenza and dysregulated coagulation and angiogenesis are alsofeatures25increased and dysregulated th1 and th17 responses werepresent in ards in middle eastern respiratory syndrome merscov and ‚uenza2627 the occurrence of severe lung disease at“ days postinfection dpi reflects the dual features of spreadof infection to the lrt and coincident development of adaptiveimmune responses with heightened activation of virusspecific teffector cells this coincides with lymphopenia associated withsevere disease and is a predictive marker earlier in disease withworse outcome28 there is also evidence that tcells aredysfunctional with increased expression of exhaustion moleculesrelated to heightened systemic ‚ammation29 the role ofelevated systemic immune dysregulation is supported by analysisof the transcriptomic responses in people with sarscov2infection demonstrating that impaired interferon ifn responseswere related to persistent viremia and increased systemicil6 and il1030 elevated‚ammation with elevated tnfαsystemic levels of il17 are also present in critical illness with ardsand heightened ‚ammation and it is plausible that increasedth17 responses drive ongoing ‚ammation31 while interestingthese are observations often performed with limited patientnumbers where ards and disease severity are associated withheightened ‚ammatory and immune activation and a causativerole cannot be established2732 interrogation of representativeanimal models is needed to define cause and effect elucidatemechanisms of pathogenesis and test treatmentssmall animal modelsa range of animal models have been used to study covid19 withvarying susceptibility likely dependent on species specific makeup for ace2 fig fig ace2 protein phylogenetic divergence and in vivo modelseverity left panel fast minimum evolution distance tree for ace2protein sequences using griphin for evolutionary distance unsortedspecies included are gallus gallus chicken anas platyrhynchos duckcavia porcellus guinea pig mustela putoris furo ferret canis lupusfamiliaris dog felis catus cat sus scrofa pig rousettus aegyptiacusfruitbat mesocricetus auratus golden hamster mus musculus mousecallithrixjacchus common marmoset macaca mulatta rhesus macaque macaca fascicularis cynomolgus macaque chlorocebus aethiopsafrican green monkey and homo sapiens macaques are representedas one image due to close divergence severity of disease is colorcoded from refractory to infection blue no virus detected to severered shedding common marmoset and guinea pig have only beenassessed for sarscov all others with sarscov2 scale indicates amino acid divergencemouse modelssmall animal models are widely used to study emerging virusesbut often they need to be genetically modified or the virus needsto be adapted for different species to be susceptible and this is thecase for sarscov2 in most studies to date the mouse strainshave been incompletely or incorrectly described and should beincluded in future publicationsgenetically modified miceinbred mouse strains were used to model sarscovseveralinfection including balbc33 c57bl634 and œ129svev incorrectlyreported name35 as well as factordeficient ˆ’ˆ’ mice such ascd1ˆ’ˆ’ rag1ˆ’ˆ’ and stat1ˆ’ˆ’“ the identification of ace2 asthe host receptor for sarscov38 initiated considerable globalinterest in developing murine models that are representative ofhuman disease this led to the generation of transgenic mice thatexpress human hace2 k18hace2 in the epithelia of tissues andans including lungs liver kidney spleen heart and gut k18hace2 mice still express the mouse mace2 however this isprimarily localised in the ileum39 they are highly susceptible tosarscov infection and experience high virallung titressignificant weight loss and morbidity from dpi39 viral dissemination to the brain was the main cause of death40 similar resultswere obtained in another transgenic strain expressing hace2under the control of the mace2 promoter41mucosal immunology __ 0cwith the mers outbreak in and identification of that virus™entry receptor dipeptidyl peptidase4 dpp4 cd26 it was foundthat neither wildtype wt nor immunodeficient mice weresusceptible to merscov infection42 transgenic mice expressinghuman hdpp4 in epithelial and endothelial cells in the lungbrain heart liver kidney spleen and intestine were generated43they were highly susceptible to merscov infection withsignificant weight loss high virallung titres and ‚ammatorycytokine production from dpi and mortality from day inother studies exons “ of the mdpp4 locus were modified toresemble hdpp4 expression44“ which led to merscov lungreplication but not disease development4748with the discovery of sarscov2ace2 interactions21249 globalinterest in hace2 mouse models reemerged however due tofalling interest with the resolution of the sars outbreak most labsceased maintaining hace2 mice to satisfy the global demand forhace2 mice the jackson laboratory reanimated k18hace2 micewhich are becoming available the lagtime has slowed theunderstanding of disease mechanisms in covid19 and theidentification of effective drug and vaccine candidates to progressto clinical trialsthe first live sarscov2 infection model used transgenic miceexpressing hace2 under the control of the mace2 promoter4150mice had significant weight loss over a 14day infection periodand high viral lung titres “ dpi histological lung examinationrevealed moderate interstitial pneumonia ltration of lymphocytes mucus accumulation and desquamation of bronchoepithelial cells from day there were no detectable viral titres orpathology in other tissues or ans except on day in theintestine suggesting that infection is localized almost exclusivelyto the lungssimilarly transgenic mice overexpressing hace2 under thecontrol of hfh4foxj1 lung ciliated epithelial cellspecificpromoters are also susceptible to sarscov2 infection5152 mostinfected hfh4hace2 mice had minimal weight loss over 7days ofinfection however mice that later became moribund showedsignificant weight loss from day and significant lymphopeniaand neutrophilia in peripheral blood at day which recapitulatessevere human disease2831 lung histology showed initial macrophage and lymphocyte ltration and fibrin exudation from dpiwhich steadily progressed to severe pneumonia blockage ofterminal bronchioles extensive fibroplasia and alveolar necrosisby day in contrastlung tissuespecificity50 hfh4hace2 infected mice had detectable viral titresin the lung eyes brain and heart suggesting that the virus mayhave additional tissue tropisms following initial lung infection5051reinfection following recovery from initial sarscov2 infectionresulted in reduced weight loss and viral titres and improvedsurvivalindicating the development of protective immunityfollowing initial challengeto previous findings ofrecentlythe first k18hace2 sarscov2 infection wasexamined53 mice exhibited no clinical symptoms or weight lossuntil dpi by day mice had weight loss with variableclinical presentation ranging from reduced activity to increasedrespiration and lethargy infected mice also had moderate virallung titres suggesting productive infection bronchoalveolarlavage revealed increased ltrating granulocytes monocytesand eosinophils accompanied by alveolar debris and septalthickening on histopathology analysis while these results areencouraging the study only examined five mice up until dpi inagreement with these initial finding53 a recent preprint papersimilarly shows that sarscov2 infected k18hace2 mice lost“ weight by dpi which steadily decreased to bodyweight by dpi54 they had high virallung titres from dpiaccompanied by modest viral titres in the heart brain kidney andspleen declines in pulmonary function were notable at dpievidenced by reduced inspiratory capacity and increased tissueresistance and elastance rnasequencing analysis of infectedmucosal immunology __animal and translational models of sarscov2 infection and covid19md johansen type i and iiinnate immunelung tissues identified the upregulation ofsignatures particularly nfkbdependentifnsignalling and leucocyte activation pathways another preprintpaper shows that sarscov2 infected k18hace2 mice produce arobust th1217 cytokine storm in the lungs and spleens from dpi55 highlighting the relevance of this mouse model thatrecapitulates critical human clinical features of covid19 importantly multiple reports have shown that sarscov2 infection offrom dpi55“k18hace2 mice is dosedependently fatalsuggesting that it is similarly lethal as sarscov39 in thesetransgenic miceadenoviral systemsadenovirus vectorbased systems can be used to insert humanreceptors into mouse genomes and are valuable for use withalready factordeficient or transgenic mice replicationdefectiveadenovirus vectors have been used to insert hddp4 into wt micerendering them susceptible to merscov infection58 infectedmice developed pneumonia with extensive pulmonary immunecell ltration and viral clearance by dpi58 but were less affectedthan fully hdpp4 transgenic micesimilarly transduction of balbc mice with adenovirus containing hace2 advhace2 led to stable hace2 expression in thelungs from h posttransfection59 sarscov2 infected advhace2 mice had weight loss over days high viral lungtitres and modest titres in the heart brainliver and spleenextensive neutrophil accumulation in perivascular and alveolarlocations and vascular congestion upon histological examinationadministration of antiifnar1 monoclonal antibodies to transiently inhibit typei ifn signalling resulted in up to weight lossand more severe lung ‚ammation compared to infection alonein this system neutralizing antibodies against sarscov2 sprotein 1b07 were protective against severe disease mice lostless body weight and had lower viral titres in the lung heart andspleen at dpi and reduced expression of pro‚ammatorycytokines and chemokines ifnb il6 cxcl10 cxcl1 ccl2 ccl5 andimmune cell ltrates in the lungs at dpicrispr systemsusing crisprcas9 an alternative humanised hace2 mousemodel was developed where the mace2 was disrupted byinserting hace2 linked to the red fluorescent protein tdtomato60hace2 expression is under the control of the mace2 promoter inthe native locus with expression predominantly in the lungs andkidneys similar to mace2 in wt mice sarscov2 infectedhumanized hace2 mice had high viral titres in the brain tracheaand lungs with no differences between young and aged micehowever infected aged mice had lung neutrophilia and extensivealveolarinjury and focal hemorrhagingcompared to young mice intragastric infection resulted in highviral titres in the trachea and lungs suggesting that the oraladministration route can lead to productive pulmonary infectionthickening vascularmouseadapted virusesviruses can be adapted to infect wt mice through serial passagingor targeted mutation their use is advantageous as they reducebiological risks to researchers and may more closely resemblenatural hostpathogen interactions in mice however they arelimited due to mouse adaptation that may result in infection thatdoes not recapitulate all aspects of human diseasethis approach was applied to sarscov which was seriallypassaged in the respiratory tracts of young balbc mice this ledto the generation of a mouseadapted sarscov strain ma15which was lethal to mice from dpi61 infection resulted in highviral titres in the lungs from dpifollowed by viremia anddiffusion to extrapulmonary sites including the brain liver andspleen significant lymphopenia and neutrophilia mild and focalpneumonitis and necrotic cellular debris in the airways and alveoli 0canimal and translational models of sarscov2 infection and covid19md johansen another mouseadapted sarscov strain v2163 was producedby serial passage in 6weekold balbc mice62 infection resultedin more severe symptoms and higher mortality rate than withma15 with greaterimmune responses and lung pathologymouseadapted sarscov strains lacking the critical viral envelopei protein induce varying degrees of protection against reinfectionwith virulent strains highlighting the potential for liveattenuatedvaccines6364serial passage of merscov through the lungs of mice withhuman modified exons “ of dpp4 resulted in a virus thatinduced significant weight loss and mice became moribund from dpi mice had high stable viral titres in the lungs and blood‚ammatory cellltrates and oedema necrotic debris andvascular permeabilization in lungs47mouseadapted sarscov2 has been reported in a preprint withmutations in the receptor binding domain rbd of the spike proteinfollowing serial passageinducing productive infection of bothyoung and aged wt balbc mice65 infected mice had high virallung loads up to dpi and displayed mild pneumonia with‚ammatory cell ltration alveolar damage focal exudation andhaemorrhage and endothelial cell denaturation the efficacy of arbdfc based vaccine was examined in this model which inducedthe production of neutralizing antibodies which potently inhibitedthe infection a similar preprint describes the modification of therbd of the sarscov2 s protein which facilitated the efficientbinding of the s protein to mace2 for host cell entry66 infectionwith this virus resulted in viral replication in the upper and lowerairways of young and aged balbc mice aged mice had greaterweight loss and pulmonary function decline compared to youngmice reproducing important aspects of human diseasea summary of the different mouse models that are permissiveto sarscov2 infection table and a comparison of featuresbetween mouse models and human covid19 fig are showncovid191875“ animal models that combine these risk factorswith infection will be invaluable in elucidating mechanisms ofincreased susceptibility and severity8081chronic respiratory diseasesboth cigarette smoking cs and copd are strong independentrisk factors for severe covid19 with extensive lung damage andincreased mortality82 cs upregulates ace2 expression in theairways which may increase infection risk83 the use of shorttermmurine models thatreplicate csinduced copd has greatlyincreased our understanding of disease and identified and testednovel therapeutic interventions808184“early data from china showed asthma prevalence in covid19patients were lower than the general population suggesting thatasthma may be protective89 however emerging reports showthat asthma is one ofthe most prevalent comorbidities inhospitalized covid19 patients90 and is associated with higher riskof death especially in severe asthma based on oral corticosteroiduse21 elucidating how severe asthma predisposes to severecovid19 is needed and can be achieved using preclinical animalmodels of severe asthma that recapitulate the human disease91“k18hace2 mice and sarscov2 infectionlung fibrosis is the most severe sequelae of covid19 in up to of the patients after months94“ the mechanisms areunclear but ‚ammation and cytokine storm likely contribute7sarscov2 infection of human alveolar epithelial cells results inaltered profibrotic gene expression similar to pulmonary fibrosisincluding ace2 tgfβ connective tissue growth factor tissueinhibitor of metalloproteinase3 and fibronectin98 fibrotic sequalae can be assessed in mouse models with long rest periods afterinfection8699 bleomycininduced experimental pulmonary fibrosiscould be combined with sarscov2 infection to investigatesequalae8699diversified modelsthe immediate aim of producing infectionpermissive mice ormouseadapted viruses is to generate models with high respiratory viral titres and lung lesions resembling those observed inhumans host factors are associated with increased risk of severecomplications including age and obesity67 and comorbidities likecopd cvd and diabetes68 human genetic variantslikelymodulate susceptibility to covid1969in mice the impact of host genetic variations were investigatedin sarscov using the collaborative cross cc a collection ofmouse inbred strains produced by crossing eight founder inbredstrainsincluding five classic laboratory strains aj c57bl6j129s1svimj nodshiltj and nzohiltj and three wildderivedcasteij pwkphj and wsbeij strains70 cc strains segregate million polymorphisms and have more genetic diversity thanthe human population71 this resource is ideally suited forinvestigating the role of host genetic variants in the pathophysiology of infectious diseases72 ma15 infected cc mice had abroad range of phenotypes including weight loss and increasedlung viral titres and lung pathology73 susceptibility varied fromabsence of symptoms to mortality with normallungparenchyma and lung viral titres at dpi that varied over logunits genetic analysis of cc strains identified trim55 and ticam2as host susceptibility genes7374 this shows that the geneticbackground of mice is important in replicating distinct humanclinical presentations investigating multiple genetic backgroundsis possible using adenovirustransduced hace2 or mouseadaptedvirusespredisposing risk factors and mouse modelschronic diseases like copd asthma lung fibrosis cvd diabetesmellitus as well as obesity male sex and old age are associatedincreased susceptibility to sarscov2 infection and severeobesity diabetes and cvdmouse models of genetic or dietary modifications to inducefeatures of human disease are widely available8687100101 inbredmouse strains such as c57blb6 have genetic susceptibility toobesity and diabetes while male mice and rats are more pronecompared to females101“ ace2 is highly expressed in hearttissues and so it is expected that cvd models willincreasedsusceptibility to sarscov2 infection104 the link between type andor diabetes and ace2 expression is unknown withconflicting reports on the levels of expression in diabetesprogression and severity105106 there are numerous mousemodels of type and diabetes that are chemically or geneticallyinduced and for type diabetes typically incorporate obesity107sarscov and sarscov2 infection has not yet been explored inthese models but these mice will likely be highly susceptible toinfection with pulmonary decline and increased mortality asobserved in humansagerelated susceptibilityold age is the main risk factor for severe covid19108 which isattributed to comorbidityimmunosenescence malnutritionresidential care and biological aging changes109 ace2 expressionin human lungs may either be unchanged83 or decreased104 withold age while expression is increased in the olfactory epitheliumwhich may contribute to susceptibility to sarscov2 infection110the significance of age is exemplified in murine models whereinfection of young balbc and c57blb6 mice with sarscovresulted in high viral titres in the upper and lower airways butwith no disease or mortality33 however aged balbc mice hadsignificant weight loss and severe disease with extensive alveolardamage and bronchiolitis111 ace2 levels are decreased in agedcompared to younger mice which may explain differences indisease severity112 itis unknown whether aged mice aresusceptible to sarscov2 infection however evidence frommucosal immunology __ 0cybsaytilatrommrofinuhtiwipdybssolithgewllewsaipdmorfgnulehtnisertitlarivhghiipdinarbdnasetanbrutilasanehtnisertitlarivetaredomdnasgnulnidevresbomrotsienkotycipd“morfipdmorfneepslsgnulehtnisertitlarivhghiipdybssolithgewmorfyticapacyrotaripserdecuderipdmorfneepsldnayendkiinarbtraehehtnisertitlarivtsedomdnasyawhtapesnopserenummietanniroflnoitaugerpuhghiswohseussitgnuldetcefnifoqesanripdlygoohtapotsihufp×ufp×ecnereferemoctuoesaesidsesodsuoitcefninoitasilacoldnanoisserpxeecahretomorpenilesuomlarivhghiderevocerllatubithgewydobtsoliytidbromonsngislacinilcisuovboongnulehtniecimsertitdcti×otpuyendkidnaenitsetnininoisserpxehghitraehninoisserpxeetaredomsgnulninoisserpxewolretomorpecambhecahsisenegohtapvocsrasienmaxeotdesusledomesuomeballiavafoyrammuselbatdnanoitammaflniinarbdnagnulevisnetxeipdybsertitgnullarivhghiipdybssolithgewufp×otpuinarbninoisserpxeegamadgnulerevesdnaailihportuendahecimdnubiromhghiipdybesaesidcitamotpmysdnassolithgewlygoohtapotsihybdecnedveisgnulnistnuoclygoohtapotsihllecdnayrotammaflninoitammaflnidnasertitlarivgnulevisnetxesyendkidnarevilninoisserpxewoljxofdcti×woltraehdnarevilyendkineepslenitsetnillamsninoisserpxeetaredomnoocldnasgnulninoisserpxehghiknitarekotycecahkdedidnaithgewydobtsolecimfonoitroporpufp×inarbdnatugsgnulninoisserpxehghihfhrotcafnoitpircsnartdaehkrofecahhfhanimal and translational models of sarscov2 infection and covid19md johansen gnullarivhghisyadrevoithgewydobtsolytilatromdetroperondetroperecimsertituffninoisserpxesgnulninoisserpxedetropertonseussithghirehtolsurivoagemotycfolortnoctnetepmocnirednuhfhniretomorpecahvdadegagnuldnaaehcartinarbnisertitlarivhghdahisngislacinilcisuovboonithgewydobtsolecimecimufp×ninoisserpxewolyendkidnaneepsltraehdnayravoinarbenitsetnillamssgnulninoisserpxehghiretomorpecamevitanecadesinamuhrotcevlarivonedamucosal immunology __ 0canimal and translational models of sarscov2 infection and covid19md johansen fig comparison of disease features shared between humans with covid19 and mouse models of sarscov2 both humans and micedisplay similar clinical signs such as weight loss and pneumonia severe infections are often associated with increased pro‚ammatorycytokine production accompanied by high viral lung titres which correlates with extensive lung damage and significant pulmonary declinehumanised ace2 mice shows that aged mice display greaterdisease severity60 aged mice showed a reduced response toexperimental vaccination for sarscov with lowerlevels ofneutralizing antibodies than young mice113 similar to responsesto many vaccines in older people thus studying aged mice islikely to be pivotal for developing treatments and vaccines forolder peoplewas less efficient with low levels of viral antigen detectable innasal washes from two of six ferrets and infection was mild withno changes in body temperature overall the ferret model ofsarscov2 is limited as the infection is mild with little lrtinvolvement and no reported characteristics of severe disease inhumans such as oedema or ards they may provide a model tostudy sarscov2 transmissionferretsferrets have been extensively used to study ‚uenza a virus iavpathogenesis and transmission since their respiratory tract isanatomically comparable to humans114 ace2 is most abundantlyexpressed on typeii alveolar and glandular epithelial cells in thetrachea and bronchi of ferrets115 ferrets are susceptible to sarscov infection with replication in the urt and lrt increased bodytemperature sneezing and alveolar damage115“recently ferret ace2 was shown to contain the critical residuesrequired for binding by sarscov2 rbd118 the animals weresusceptible to sarscov2 infection with viral replication in theurt however only low levels of virus were detected inlungs119120 viral loads in the lung and nasal turbinates peakedat dpi with clearance by and dpi respectively whileinfectious virus was not detected outside the respiratory tract viralrna was found in the intestine saliva urine rectal swabs andfaeces up to 8dpi this suggests that sarscov2 preferentiallyreplicates in the urt of ferrets in line with this disease is mildwith reduced activity and occasional cough from days “ in moststudies elevated body temperatures are observed from dpireturning to baseline by day with no change in bodyweight119121 shi 120 reported only out of ferretsdeveloped fever and loss of appetite following intranasalinoculation with different sarscov2 isolates suggesting isolatevariability and dose may alter disease outcomes limited studieshave examined immune responses to sarscov2 in ferretsintranasal infection induced serum neutralizing antibodies119121another study compared transcriptional responses in cells fromiav and sarscov2infected ferrets122 thenasal washes ofmagnitude of antiviral and ifn responses were higher with iavcompared to sarscov2 infection with unique enrichment forcell death and leucocyte activation in the latter kim 119 usedthe ferret model to study sarscov2 transmission viral rna wasdetectable in nasal washes from ferrets h after direct contactwith intranasally inoculated animals suggesting transmission israpid and can be transmitted prior to the peak of disease dpi allsix ferrets that had direct contact developed elevated bodytemperatures in contrast to direct contact airborne transmissiongolden hamstersyrian golden hamsters have been used to model a broad rangeof viral infections123 and genetically modified animals have beengenerated124“ hamsters are susceptible to sarscov infectionwith comparable viral replication in the urt and lrt but noclinical signs of disease other than reduce activity111127128in silico structural analysis predicts that sarscov2 s proteinrbd effectively binds hamster ace2129130 infection of hamsterswith sarscov2 resulted in clinical signs such as ruffled fur rapidbreathing and weight loss from day with recovery by “dpi129 high levels of infectious virus were detected in the lungand nasal turbinates on days and and significant histologicalchanges were observed including proteinrich lung fluid exudatemononuclear cell ltration severe cell death and haemorrhageand alveolar damage transmission studies revealed efficient virusspread via direct contact between cohoused infected hamsters129 infectious virus was detectable in the lungs and nasalturbinates accompanied by lung histology changes cohousedhamsters did not lose significant body weight suggesting theinfection was less severe than with intranasally inoculation likelydue to differences in inoculum dose this suggests that sarscov infection of hamsters is not dissimilar to humans and may be amodel to study pathogenesis and transmission and test potentialtherapeuticsnonhuman primates nhpsnhp experiments account for only of all animal researchnevertheless clinical translation of nhp studies is much greaterthan for other animal models as they are closest to humanp
0
"Proteasomes are found in both the cell nucleus and cytoplasm and play a major role in the ubiquitindependent and independent nonlysosomal pathways of intracellular protein degradation Proteasomes are alsoinvolved in the turnover of various regulatory proteins antigen processing cell differentiation and apoptosis Todetermine the diagnostic value of serum proteasome in antineutrophil cytoplasmic antibody ANCAassociatedvasculitis AAV we investigated patients with AAV at various stages of the diseaseMethods Serum 20Sproteasome was measured by ELISA in patients with MPOANCAassociated microscopicpolyangiitis MPA and renal involvement Thirty of the patients provided serum samples before the initialtreatment and provided samples during remission provided samples at both time pointsResults The mean serum 20Sproteasome level was significantly higher in the activevasculitis patients ± ngmL n compared to the inactivevasculitis patients ± ngmL n p and controls ± ngmL p There were significant positive correlations between the serum 20Sproteasome level and the Birmingham Vasculitis Activity Score BVAS r p the ANCA titer r p the white blood cell WBC count r p the platelet count r p and the serum Creactive protein CRP level r p There were significant negative correlationsbetween the serum 20Sproteasome level and both the hemoglobin concentration r ˆ’ p and theserum albumin level r ˆ’ p In a multiple regression analysis there was a significant positivecorrelation between the serum 20Sproteasome level and only the BVAS results p In a receiveroperating curve analysis the area under the curve for the serum 20Sproteasome level was which is higherthan those of the WBC count and the serum CRP level Conclusion The serum level of 20Sproteasome may be a useful marker for disease activity in AAVKeywords ANCAassociated vasculitis 20Sproteasome Disease activity Microscopic polyangiitis Proteasome Correspondence khiratokyomedacjp1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki JapanFull 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 0cMaruyama BMC Rheumatology Page of BackgroundProteasomes are located in both the nucleus and cytoplasm of cells and they play a major role in theubiquitindependent and ubiquitinindependent nonlysosomal pathways of intracellular protein degradation[ ] Proteasomes are also involved in the turnover ofvarious regulatory proteins eg ratelimiting enzymes[] and proteins for cellcycle control [] or transcriptional regulation [] antigen processing [] cell differentiation [] and apoptosis [] The 26S proteasome is amulticatalytic enzyme with a highly ordered structurecomposed of at least different subunits arranged intwo subcomplexes a 20S core and a 19S regulator p [] The 20Sproteasome is composed of four ringsof nonidentical subunits two rings are composed ofseven alpha subunits and the other two rings are composed of seven beta subunits Three of the seven betasubunits have proteolytic sites the 1 2 and 5 subunits are associated with caspaselike trypsinlike andchymotrypsinlike activities respectively [] These 12 and 5 subunits cleave peptide bonds at postacidicˆ’basic and hydrophobic amino acid residues respectively [] However subunits 1 2 and 5 could be replaced with 1i 2i and 5i by interferongamma IFNÎ and this IFNÎinducible proteasome isotype is calledthe immunoproteasome []The serum proteasome levels of patients with malignanttumors are elevated because the proteasome is overexpressed in tumor cells In patients with multiple myelomaserum proteasome concentrations have been shown to beassociated with disease severity and activity [] theserum proteasome concentrations were significantly elevated in patients with multiple myeloma compared tocontrols in multiple myeloma versus monoclonal gammopathies of undetermined significance MGUS and in active versus smoldering multiple myeloma [] Similarlyelevated serum proteasome levels were also reported inautoimmune diseases characterized by Bcell abnormality[] In the present study to determine the diagnosticvalue of the serum proteasome concentration in antineutrophil cytoplasmic antibody ANCAassociated vasculitisAAV we investigated patients with myeloperoxidaseMPOAAV at various stages of the diseasePatientsPatients and controlsWe analyzed the cases of patients with MPOANCAassociated microscopic polyangiitis MPA and renal involvement The diagnosis of MPA was based on theEuropean Medicines Agency algorithm [] and patientswith other types of systemic vasculitis including eosinophlic granuromatosis with polyangiitis granulomatosis with polyangiitis and antiglomerular basementdisease were excludedOf the MPOAAV patients provided serumsamples before the initial treatment and providedsamples during remission provided samples both before the initial treatment and during remission The Birmingham Vasculitis Activity Score BVAS was used toevaluate patients™ disease activity and remission was defined as a BVAS of As controls healthy volunteersand patients with chronic kidney disease CKD wereinvestigated The causes of CKD were nephrosclerosisn chronic glomerulonephritis n diabeticnephrosclerosis n and autosomal dominant polycystic kidney disease n Sample collection and analysisThe serum samples measured by a commerciallyavailable enzymelinked immunosorbent assay ELISAkit Enzo Life Science Plymouth Meeting PA USin duplicate In brief 96well microtiter plates werecoated with a mouse anti20Sproteasome alpha6subunit monoclonal antibody and left overnight at °C followed by blocking with phosphatebuffered saline PBS containing bovine serum albumin for h atroom temperature RT A serum sample was thenadded to each well and the plates were incubated for h at RT A rabbit anti20Sproteasome polyclonalantibody was then added to each well and the plateswere incubated for h at RT followed by incubationwith a horseradishperoxidaselabeled goat antirabbitIgG antibody for h at RT The plates were finallyincubated with chromogen tetramethylbenzidine andhydrogen peroxide for min at RT and then addedwith N hydrochloride acid solutionBetween these steps the plates were washed five timeswith Trisbuffered saline The plates were immediatelyread on a microplate reader Sunrise Remote® TecanJapan Kanagawa Japan set at nm with nm as areference wavelength The inter and intraassay variationswere Statistical analysesAll statistical analyses were performed using PASW Statistics software ver IBM Japan Tokyo for Windows The data are expressed as means ± standarddeviations or as numbers with percentages of the totalThe chisquare test with Yates™ continuity correctionand Fisher™s exact test were used for differences in categorical variables and posthoc comparisons Bonferronicorrection were performed to detect differences amongthree groups The MannWhitney Utest was used fortwogroup comparisons and we conducted an analysisof variance ANOVA to assess differences among threeor more groups posthoc comparisons were made usingthe BonferroniDunn test Correlations were determinedusing Spearman™s univariate correlation test and a linear 0cMaruyama BMC Rheumatology Page of regression analysis The multiple linear regression analysis included the covariates shown to be significantly associated with the serum 20Sproteasome levelin thecorrelation analysis and the data are expressed as standardized regression coefficients We applied comparative receiveroperatingcharacteristic ROC curvesand the area under the curve AUC to assess the diseaseactivity accuracy of the the serum 20Sproteasome leveland inflammatory variables Pvalues were accepted assignificant at but in the comparisons of three ormore groups the critical pvalue was divided by thenumber of comparisons being madeResultsThe subjects™ characteristicsThe characteristics clinical symptoms and laboratorydata among the subjects of the three groups the activevasculitis patients the inactivevasculitis patients andthe controls are shown in Table At the testing therewas no patients treated with any immunosuppressant inboth active and inactive vasculitis but allinactivevasculitis patients had treated with corticosteroids dosesof prednisolone ± mgdaySerum 20Sproteasome levelsAs illustrated in Fig the mean level of serum proteasome in the activevasculitis patients ± ngmL was significantly higher than those in the inactivevasculitis patients ± ngmL p andthe controls ± ngmL p There weresignificant positive correlations between the serum 20Sproteasome levels and the BVAS results r p the MPOANCA titers r p theWBC counts r p the platelet countsTable Characteristics of subjectsAge yearsGender malefemaleBirmingham vasculitis activity scoreClinical symptomsFeverWeight lossArthralgiaEpiscleritis or uvitisSinusitisHearing lossAlveolar hemorrhageInterstitial lung diseaseArrhythmiaPericarditisHeart failureRapidly progressive glomerulonephritisPeripheral nerve damageLaboratory dataANCA titer UmLWhite blood cell mm3Hemoglobin conc gdLPlatelet count 104mm3Serum albumin gdLSerum creatinine mgdLSerum Creactive protein mgdLSerum 20Sproteasome mgdLDoses of prednisolone mg daily P vs Inactivevasculitis P vs ControlsMPOANCA associated vasculitisActivevasculitis n ± Inactivevasculitis n ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Controlsn ± ± ± ± ± ± ± ± 0cMaruyama BMC Rheumatology Page of Fig The serum levels of 20Sproteasome Closed circles means bars standard deviations circles the values for individual patientsr p and the serum CRP levels r p There were significant negative correlations between the serum 20Sproteasome levels and boththe hemoglobin concentrations r ˆ’ p and the serum albumin levels r ˆ’ p In the multiple regression analysis there was a significant positive correlation between the serum 20Sproteasome levels and only the BVAS results p Table In the activevasculitis patients there was no associationbetween the serum 20S proteasome levels and clinicalsymptoms except for pulmonary involvement Supplementary file S1 The mean serum 20S proteasome level inthe activevasculitis patients with interstitial lung diseasen ± ngmL was significantly higherthan those in the activevasculitis patients withoutpulmonary involvement n ± ngmLp and those in activevasculitis patients withalveolar hemorrhage n ± ngmL p There was no association between the serum 20Sproteasome levels and the percentages of crescent formation renal histological classification Berden™s classification [] or renal symptoms patients with chanceproteinuriahematuria and patients with rapidly progressive glomerulonephritisThe diagnostic potential for disease activityThe optimum cutoff levels for the disease activity ofvasculitis were identified from the ROC curves for theWBC count 7250mm3 serum CRP level mgdL and serum 20Sproteasome level ngmLFig The area under the curve AUC for the serumTable Correlation between the serum 20Sproteasome level and clinical parametersAgeBirmingham Vasculitis Activity ScoreANCA titerWhite blood cellHemoglobin concPlatelet countSerum albuminSerum creatinineSerum Creactive proteinUnivariate analysisrˆ’ˆ’Pvalue Multivariate analysis“ˆ’ˆ’ˆ’“ˆ’Pvalue““ 0cMaruyama BMC Rheumatology Page of polymyositis serum proteasome levels were correlatedwith serum creatinine kinase levels and serum proteasome levels were associated with disease activity [] Inthe present study elevated serum 20Sproteasome levelswere also demonstrated in patients with AAV Althoughthere was no relationship between the MPOANCA titersand the serum 20Sproteasome levels these elevationswere associated with disease activity ie the BVASTherefore the serum level of 20Sproteasome may be auseful marker for disease activity in AAVThe mechanisms that underlie the elevated serum proteasome observed in patients with AAV are not yetknown Several serum biomarkers are filtrated at theglomerulus and reabsorbed and catabolized by proximaltubular cells and the serum levels of such biomarkers inpatients with renal insufficiency are elevated due to lowurinary filtration Because we found no relationship between serum 20Sproteasome levels and serum creatinine in AAV patients we conclude that elevated serumproteasome is not associated with renal functionIn a previous investigationthe serum proteasomelevels in patients with multiple myeloma were elevateddue to overexpression in tumor cells but the mechanisms underlying these elevations in autoimmune diseases were not clarified [] On the other hand thestructure and function of serum proteasome in healthydonors and patients with autoimmune diseases SLE andRA were maintained in the same manner as the intracellular forms [] However rings of proteasomes inthe serum of patients with autoimmune diseases weredifferent from those in healthy donors and those ringscontained immunosubunits 2i and 5i [] Consideringthat proteasomes from nonimmunocompetent cells donot contain immunosubunits [] it was speculated thatserum proteasome in patients with autoimmune diseasesmay have its fraction structure added by an immunocompetent cell origin that is different from that in normal individuals Therefore the elevated serum proteasome levelsin AAV may also be associated with the activation of immunocompetent cells Further investigations are neededto clarify the mechanism by which the proteasome isreleased into the circulationBortezomib a proteasome inhibitor prevents the degradation of proteins marked by ubiquitination by inhibiting the 26S proteasome [] The main effects ofbortezomib are NFκB inhibition inhibition of cell proliferation by the stabilization of cyclindependent kinasesFig The comparative ROC curves for three measurements ofdisease activity Solid line serum levels of 20Sproteasome Dashdotted line WBC counts Dashed line serum CRP levels Dotted linereference line20Sproteasome level was which is higher thanthose of the WBC count and the serum CRP level On the ROC curve the serum 20Sproteasomehad sensitivity and specificity for the diseaseactivity Although the specificity of the serum 20Sproteasome level was less than that of the serum CRPlevelserum 20Sproteasome level was superior to that of the serumCRP level Table sensitivity ofthetheDiscussionPrevious studies have demonstrated that the serum20Sproteasome levels are elevated in individuals with autoimmune diseases In patients with various autoimmune diseases including systemic lupus erythematosusSLE polymyositis Sjögren™s syndrome antiphospholipidsyndrome rheumatoid arthritis RA systemic sclerosisautoimmune hepatitis and myasthenia gravis the serumproteasome levels were higher than in the controls[] The levels were especially and significantly high inthe patients with SLE polymyositis Sjögren™s syndromeRA and autoimmune hepatitis [] In patients withWhite blood cell countTable Comparative ROC curves for parameters of disease activity confidence interval“““Area under the curveSerum Creactive proteinSerum 20SproteasomePvalue Optimal cutoff levelsSensitivity Specificity 0cMaruyama BMC Rheumatology Page of the induction of apoptosis by the activation of cJunNH2terminal kinase the stabilization of proapoptotic proteinsand transcription factors and tumor suppressors and theinduction of cell death by activation of the terminalunfolded protein response [] Bortezomib has been approved for clinical use in patients with multiple myelomaand bortezomib treatment has implications for antibodymediated immune diseases as well []The efficacy of bortezomib was demonstrated in amouse model of MPOANCAassociated glomerulonephritis [] That is in antiMPOassociated glomerulonephritisinduced by immunizing MPOdeficientmice with murine MPO followed by irradiation and thetransplantation of wildtype bone marrow proteinuriaalbuminuria and hematuria were significantly reducedcompared to the controls by both standard steroidcyclophosphamide treatment and bortezomib treatment[] Moreover the percentage of glomeruli with crescent or necrosis formation was reduced by both treatments The clinical efficacy of bortezomib for AAV hasnot been determined because only one case of an AAVpatient treated with bortezomib was reported In thatcase complete remission could not be achieved by acombination treatment with corticosteroid cyclophosphamide and rituximab therefore bortezomib mgm2week for weeks was added [] After the additionof bortezomib the patient achieved complete remissionand the doses of corticosteroid could be withdrawn []Thusthe proteasome may be associated with thedevelopment of AAV and inhibition of the proteasomemay be effective for inducing the remission of AAVOur study has several limitations The study population was small and limited to MPOAAV patients withrenal involvement and thus further studies are neededto compare patients with PR3AAV or nonrenal vasculitis In addition this was a retrospective crosssectionalstudy a larger prospective longitudinal study includingvasculitis patients with relapse would provide more definitive results Since the present study was performed atone facility it is necessary to verify the accuracy of theELISA test Moreover allinactivevasculitis patientswere treated with corticosteroids at the testing so treatments themselves may affected to decreased levels in inactive vasculitis Therefore further studies are needed tocompare AAV patients without treatments at the testingor to investigate other diseases patients treated withwithout corticosteroids Finally although we did demonstrate that serum 20Sproteasome levels were elevated inour patients with AAV the cause of this elevation wasnot identified In patients with multiple myeloma elevation of serum 20Sproteasome may be associated withoverexpression in tumor cells or abnormal cellular turnover [] On the other hand elevation of serum 20Sproteasome was observed in septic patients and therelation between elevated serum 20Sproteasome levelsand increased lymphocyte apoptosis was demonstratedin critically ill patients [] In patinets with RA andSLE it was speculated that the expression of inflammatory cytokines may have influenced the elevation of theserum 20Sproteasome [] Althoug elevated serum20Sproteasome in active AAV may be reflected an acutephase response there was no significant correlation between the serum 20Sproteasome levels and serum CRPlevels in the multiple regression analysis To clarify themechanisms of the serum 20Sproteasome elevation invasculitis patients further in vitro and ex vivo investigations are neededConclusionThe serum levels of 20Sproteasome in our patients withactive MPOAAV were significantly elevated compared tothe levels in the patients with inactive MPOAAV and thecontrols Moreover the serum levels of 20Sproteasomewere related to the BVAS results The serum level of 20Sproteasome may therefore be a useful marker for diseaseactivity in AAVSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s41927020001374Additional file Supplementary file S1 The relationships betweenthe serum 20S proteasome levels and the patients™ clinical symptomsAbbreviationsAAV Antineutrophil cytoplasmic antibodyassociated vasculitisANCA Antineutrophil cytoplasmic antibody BVAS Birmingham VasculitisActivity Score MPA Microscopic polyangiitis MPO MyeloperoxidaseAcknowledgementsPart of this study was reported at the 18th International Vasculitis and ANCAWorkshop Tokyo and it was published in Rheumatology suppl as an abstractAuthors™ contributionsHM and KH designed the study executed the experiments and participatedin the data management statistical analyses and reporting logicalinterpretation and presentation of the results MY KO and RT participated inthe data collection MT and HS took part in the logical interpretation andpresentation of the results KH and MK coanized the course of the workAll authors read and approved the final manuscriptFundingThis study was supported in part by a research grant to KH from MSD KKTokyo Japan The funds for this study were used only to purchase ELISAkitsAvailability of data and materialsAll of the raw datasets used and analyzed in this study are available uponreasonable request from the corresponding authorEthics approval and consent to participateAll procedures performed in this study involving human participants were inaccordance with the ethical standards of the institutional committee andwith the Declaration of Helsinki and its later amendments orcomparable ethical standards The study protocol was approved by the 0cMaruyama BMC Rheumatology Page of Zoeger A Blau M Egerer K Feist E Dahlmann B Circulating proteasomesare functional and have a subtype pattern distinct from 20S proteasomes inmajor blood cells Clin Chem “Froment C UttenweilerJoseph S BousquetDubouch MP Matondo MBes JP Esmenjaud C Lacroix C Monsarrat B BurletSchiltz O Aquantitative proteomic approach using twodimensional gel electrophoresisand isotopecoded affinity tag labeling for studying human 20S proteasomeheterogeneity Proteomics “ Hideshima T Richardson P Chauhan D Palombella VJ Elliott PJ Adams JAnderson KC The proteasome inhibitor PS341 inhibits growth inducesapoptosis and overcomes drug resistance in human multiple myelomacells Cancer Res “ Boccadoro M Man G Cavenagh J Preclinical evaluation of theproteasome inhibitor bortezomib in cancer therapy Cancer Cell Int Fröhlich K Holle JU Aries PM Gross WL Moosig F Successful use ofbortezomib in a patient with systemic lupus erythematosus and multiplemyeloma Ann Rheum Dis “ Bontscho J Schreiber A Manz RA Schneider W Luft FC Kettritz RMyeloperoxidasespecific plasma cell depletion by bortezomib protectsfrom antineutrophil cytoplasmic autoantibodiesinducedglomerulonephritis J Am Soc Nephrol “ Novikov P Moiseev S Bulanov N Shchegoleva E Bortezomib in refractoryANCAassociated vasculitis a new option Ann Rheum Dis 2016751e9 Yousef AA Suliman GA Mabrouk MM The value of correlation of serum 20Sproteasome concentration and percentage of lymphocytic apoptosis incritically ill patients a prospective observational study Crit Care R215Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsEthics Committees of Tokyo Medical University Ibaraki Medical CenterWritten informed consent for inclusion in the study was obtained from allpatientsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki Japan 2Department of Intensive CareMedicine Tokyo Medical University Ibaraki Medical Center Ami IbarakiJapan 3Department of Nephrology Tokyo Medical University ShinjukuTokyo JapanReceived April Accepted May ReferencesArrigo AP Tanaka K Goldberg AL Welch WJ Identity of the 19S ˜prosome™p with the large multifunctional protease complex of mammaliancells the proteasome Nature “Hershko A Ciechanover A The ubiquitin system Annu Rev Biochem “ Murakami Y Matsufuji S Kameji T Hayashi S Igarashi K Tamura T Tanaka KIchihara A Ornithine decarboxylase is degraded by the 26S proteasomewithout ubiquitination Nature “Hershko A Roles of ubiquitinmediated proteolysis in cell cycle control CurrOpin Cell Biol “Kho CJ Huggins GS Endege WO Hsieh CM Lee ME Haber E Degradationof E2A proteins through a ubiquitinconjugating enzyme UbcE2A J BiolChem “Stoltze L Nussbaum AK Sijts A Emmerich NP Kloetzel PM Schild H Thefunction of the proteasome system in MHC class I antigen processingImmunol Today “Baz A Henry L Caravano R Scherrer K Bureau JP Changes in the subunitdistribution of prosomes MCPproteasomes during the differentiation ofhuman leukemic cells Int J Cancer “Pasquini LA Marta CB Adamo AM Pasquini JM Soto EF Relationshipbetween the ubiquitindependent pathway and apoptosis in different cellsof the central nervous system effect of thyroid hormones Neurochem Res“Jung T Grune T Structure of the proteasome Prog Mol Biol Transl Sci “ Groll M Ditzel L Lowe Löwe J Stock D Bochtler M Bartunik HD Huber RStructure of 20S proteasome from yeast at a resolution Nature “ Boes B Hengel H Ruppert T Multhaup G Koszinowski UH Kloetzel PMInterferon gamma stimulation modulates the proteolytic activity andcleavage site preference of 20S mouse proteasomes J Exp Med “Jakob C Egerer K Liebisch P Türkmen S Zavrski I Kuckelkorn U Heider UKaiser M Fleissner C Sterz J Kleeberg L Feist E Burmester GR Kloetzel PMSezer O Circulating proteasome levels are an independent prognosticfactor for survival in multiple myeloma Blood “Egerer K Kuckelkorn U Rudolph PE Rückert JC Dörner T Burmester GRKloetzel PM Feist E Circulating proteasomes are markers of cell damageand immunologic activity in autoimmune diseases J Rheumatol “ Watts R Lane S Hanslik T Hauser T Hellmich B Koldingsnes W Mahr ASegelmark M CohenTervaert JW Scott D Development and validation of aconsensus methodology for the classification of the ANCAassociatedvasculitides and polyarteritis nodosa for epidemiological studies AnnRheum Dis “ Berden AE Ferrario F Hagen EC Jayne DR Jennette JC Joh K Neumann INoël LH Pusey CD Waldherr R Bruijn JA Bajema IM Histopathologicclassification of ANCAassociated glomerulonephritis J Am Soc Nephrol“ 0c"
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the bacteroides fragilis b fragilis produce biofilm for colonisation in the intestinal tract can cause a series of inflammatory reactions due to b fragilis toxin bft which can lead to chronic intestinal inflammation and tissue injury and play a crucial role leading to colorectal cancer crc the enterotoxigenic b fragilis etbf forms biofilm and produce toxin and play a role in crc whereas the nontoxigenic b fragilis ntbf does not produce toxin the etbf triggers the expression of cyclooxygenase cox2 that releases pge2 for inducing inflammation and control cell proliferation from chronic intestinal inflammation to cancer development it involves signal transducers and activators of transcription stat3 activation stat3 activates by the interaction between epithelial cells and bft thus regulatory tcell tregs will activates and reduce interleukin il2 amount as the level of il2 drops thelper th17 cells are generated leading to increase in il17 levels il17 is implicated in early intestinal inflammation and promotes cancer cell survival and proliferation and consequently triggers il6 production that activate stat3 pathway additionally bft degrades ecadherin hence alteration of signalling pathways can upregulate spermine oxidase leading to cell morphology and promote carcinogenesis and irreversible dna damage patient with familial adenomatous polyposis fap disease displays a high level of tumour load in the colon this disease is caused by germline mutation of the adenomatous polyposis coli apc gene that increases bacterial adherence to the mucosa layer mutatedapc gene genotype with etbf increases the chances of crc development therefore the colonisation of the etbf in the intestinal tract depicts tumour aetiology can result in risk of hostility and effect on human health keywords bacteroides fragilis colon cancer stat3 pathway bacteroides fragilis toxin inflammationintroductionbacteroides species are nonspore forming anaerobe and gramnegative bacteria there are more than different species of bacteroides these bacteria act as normal flora in the intestine to maintain healthy intestinal microflora in humans bacteroides fragilis b fragilis has two classes nontoxigenic b fragilis ntbf and enterotoxigenic b fragilis etbf the differences between ntbf and etbf are the presence of b fragilis toxin bft gene and its ability to produce biofilm bft product is a kda zincdependent metalloprotease toxin also known as fragilysin or bft “ bft plays an important role in intestinal inflammation and tissue injury by damaging the tight junction and increasing intestinal permeability furthermore it has been proven that tissue inflammation and injury promote cancer formation simultaneously the biofilm produced by b fragilis induces carcinogenesis fortunately only etbf encompasses bft and can produce biofilms hence ntbf does not harm the intestinal tract malays j med sci jul“aug “wwwmjmsusmmy penerbit universiti sains malaysia this work is licensed under the terms of the creative commons attribution cc by httpcreativecommonslicensesby40 0cmalays j med sci jul“aug “in the united states colorectal cancer crc is the third most common cancer in both genders it is also the second most common cancerrelated death especially for older patients who are ‰¥ years old in the american cancer society stated that there were new cases of crcs that led to the death of people moreover crc is the fourth leading cancer resulting in deaths worldwide inflammatory bowel disease ibd and genetic mutations are factors predisposing an individual towards colon cancer this indicates that crc has a high mortality rate “microbes are capable in promoting cancer development through several routes such as activation of chronic inflammation alteration of tumour microenvironment and production of toxins that damage dna when there is chronic etbf colonisation in the intestine it stimulates chronic intestinal inflammation triggering signal transducers and activators of transcription stat3 activation which contributes to interleukin il17 production il17 is involved in colon inflammation bft produced by etbf causes the alteration of signalling pathways and production of reactive oxygen species ros that leads to dna damage and cleavage of ecadherin in the below review we have provided a general information regarding bft produced by etbf triggering crc developmentliterature reviewcolon cancer associated with microbespromote nutrition in the human gastrointestinal tract there are nearly trillion microbes out of which make up normal flora in the intestine meanwhile the normal flora is characterised into beneficial and harmful microbes beneficial microbes including production of vitamins in the intestine and prevent disease formation however harmful microbes produce carcinogenic toxin and substances in the intestine these harmful substances may cause cancer there are many types of bacteria that stimulate a variety of cancer formation through their respective site of inflammation eg bacteria such as enterococcus faecalis e faecalis colibactinproducing escherichia coli e coli and etbf are involved in crc development however the mechanisms between each bacterium in contributing to crc formation are different for instance e faecalis damages the dna through ros colibactinproducing e coli produces colibactin that damages the dna and etbf produces bft that contributes to inflammation and immunecell infiltration intestinal dysbiosis inflammation and colon cancergenetic normal flora is advantageous to a person as it maintains intestinal health and gut homeostasis however as the bacteria such as etbf in the gut undergoes dysbiosis it brings harmful effects to the person according to deng et al a correlation was observed between microbiota imbalance and cancer progression while liu et al claimed is associated with that crc development intestinal microecology disorder imbalance among microbiota leads to bacterial infection that can progress to chronic inflammation one of the main environmental risk factors contributing to crc development is chronic intestinal inflammation chronic inflammation alters cellular microenvironment enhances gene mutation inhibits apoptosis and induces neovascularisation and cell proliferation that causes precancerous conditions eventually leading cancer simultaneously chronic inflammation alterations that directly affect the stat3 pathway and promoting there are three stages involved in tumour development namely initiation promotion and progression during initiation and progression cancer cells and microbes interact both producing genetic inflammatory“immunological factors that are responsible for their survival and replication in tumour progression tumour cells interact with the inflammatory cells in the tumour microenvironment these tumour cells inflammatory“immunological factors to attract the inflammatory cells and the stromal cells simultaneously activate both stromal cells start to produce various soluble factors including growth factors and protease these soluble factors play an important role in facilitating the growth differentiation and survival of tumour cells hence it promotes tumour progression and promotion additionally cytokines or microbes promote cancer by changing genetic sequence during gene mutation epithelial cells inflammatory and activated carcinogenesis cytokines chemokines causes and secrete wwwmjmsusmmy 0creplicate rapidly and develop into a hyperplastic epithelium which progresses into adenomas and then towards adenocarcinomas both adenomas and adenocarcinomas affect the growth rate of colonic epithelial cells and improve the cells™ toleration towards apoptosis and abnormal cells escape from the immune cells furthermore these invade submucosa turning into cancer when the growth of malignant cells continues the tumour continues to spread in the colon thus carcinogenesis becomes more efficientadenocarcinomas begin to ibd is an example of chronic intestinal inflammation that is associated with etbf pathogenic bacteria are capable of stimulating infection inflammation and carcinogenesis whereas the relationship between ibd and crc is well established surprisingly patients with ibd show a high level of immunoglobulin ig g antibodies il6 vascular endothelial growth factor vegf and tumour necrosis factor tnf igg antibodies are responsible for killing bacteria moving into the intestinal lumen simultaneously il6 and vegf are responsible for stat3 activation ibd is also known as ulcerative colitis uc and crohn™s disease cd this chronic intestinal inflammation increases the risk of colitisassociated crc the probability of which depends on multiple casual factors including severity duration of inflammation in the intestine and gut microbiota imbalance “ patients with uc or cd have “ folds higher incidence of crc when compared to healthy individuals it is also stated that patients with uc and cd have and respectively higher risks of crc compared to a normal healthy person this indicates that patients with uc tend to be more susceptible to crc than those with cd furthermore it is evident that the large intestine tends to have a higher risk of crc compared to the small intestine which can be attributed to the higher amount of bacteria simultaneously people with ibd and crc have a higher quantity of etbf in the intestine or stool examination compared to healthy persons additionally etbf are biofilm producers they can reduce or redistribute ecadherin in the colonic epithelial cells trigger the production of il6 by epithelial cells activate stat3 pathway and enhance cells proliferation at the site of crypt epithelial in normal colon mucosa this shows that biofilms are associated with the risk of colon cancer development review article formation of colon cancercox enzymes involved in inflammation carcinogenesis and biomarkerchronic inflammation is a principal factor that contributes to carcinogenesis prostaglandin is a paracrine hormone that plays an important role in inflammation cyclooxygenase cox is the ratelimiting enzyme responsible for producing prostaglandins cox1 and cox2 are the isoforms of cox enzymes that break down arachidonic acid into prostaglandins cox2 plays an important role in maintaining environment for the development of cancer inflammation cox2 is normally expressed in epithelial and stromal cells and the expression level is increased in both inflammation and cancer due to the presence of proinflammatory cytokines additionally bft triggers colonic epithelial cells to express cox2 but not cox1 cox2 releases prostaglandin e2 pge2 that triggers pain and inflammation at the site of tissue injury simultaneously pge2 controls cell proliferation by binding at the cell receptor and activating oncogenic signalling pathways thus it is proven that cox2 plays an important role in carcinogenesis and cancer progression by promoting cell proliferation angiogenesis and cancer stem cell formation inhibiting cell apoptosis and heightening metastatic potential through producing pge2 “the in certain studies it is stated that aspirin and nonsteroidal antiinflammatory drugs have the ability to inhibit the activity of cox enzyme which reduces inflammatory response thus it delays crc occurrence fortunately cox1 and cox2 act as biomarkers for screening purposes the biomarker is defined as any substance structure or process that is measurable in the body to determine the incidence of a disease it is commonly detected in circulation and body fluids cox1 is present in most cells thus it is not a specific biomarker however cox2 is only detected when is stimulated by trauma release of cytokines and stimulation of arachidonate metabolism by a toxin such as bft thus cox2 acts as a useful biomarker to detect “ cox2 is also a useful biomarker for colorectal carcinogenesis screening the level of cox2 biomarker in the blood is dependent upon epithelial cell proliferation apoptosis inhibition and neoangiogenesis patients with crc have high levels of cox2 compared to normal individuals “ indicating more aggressive growth rate and higher mortality rate this inflammatory responses inflammation the wwwmjmsusmmy 0cmalays j med sci jul“aug “suggests that cox2 expression is correlated to the aggressiveness of growth rate and mortality rate etbf activates stat3immune is activated to eliminate regulation of etbf is associated with ibd due to the abnormal response to bacteria the systemic adaptive immune response foreign antigens in the body this action eventually reduces intestinal mucosal tolerance although immune cells kill foreign antigens neutrophils and th17 cells contribute to inflammation and tumourigenesis transcription factors are known as stat protein family comprising seven members each stat protein responds to its specific cytokines they play an important role in regulating immune responses by controlling th cell types generation for instance the activation and generation of th17 cells require transcription factor stat3 protein the roles of stat3 protein include promotion of cell proliferation cell survival inflammation cellular transformation metastasis of cancer blood vessel formation and tumourpromoting moreover stat3 intrinsic pathway for cancer inflammation it induces genes in tumour cells that are responsible for inflammation within a tumour cell it exhibits an overly expressed stat3 pathway inflammation is a major factors etbf has the ability to activate stat3 rapidly in both colonic epithelial cells and colonic mucosal immune cells through phosphorylation and nuclear translocation however stat3 activation first occurs in colonic mucosal immune cells followed by colonic epithelial cells to activate stat3 in immune cells epithelial cells should respond in the production of cytokines such as il6 il10 and il23 besides cytokines growth including vegf and fibroblast growth factor fgf2 are also involved in activating stat3 when etbf and bft first interact with colonic epithelial cells they stimulate early stat3 activation in colonic mucosal immune cells this stat3 activation continuously rises slowly until it reaches the peak level the peak indicates that etbf activates the immune system due to barrier dysfunction during etbfinduced colitis it activates both stat3 and th17 immune response in the colonic mucosa stat3 activation induces prooncogenic inflammatory pathways and increases the permeability of mucosa although stat3 activation is longterm and lasts for months it highly increases the chance of getting a tumour as a result of chronic inflammation additionally stat3 activation promotes the accumulation of tumour regulatory tcell tregs and blocks the generation of antitumour immune responses which give an adverse effect to the body this abnormal persistent stat3 activation increases the cancer cell tolerance prevents rejection of cancer by the immune system reduces the effectiveness of immunotherapy and enhances the effectiveness of oncogenesis activated stat3 predominantly detected in human cancers is constitutively activated and depicts its association with neoplasms patients with ibd tend to show stat3 activation and a high level of th17 cells and il17 the level of activated stat3 in patients with ibd and dysplasia is different from patients with ibd and without dysplasia patients with ibd and dysplasia show a higher level of activated stat3 compared to those without dysplasia simultaneously the level of activated stat3 increases together with the continuum of dysplasia to colitisassociated cancer it is clear that b fragilis can either be toxigenic or nontoxigenic the latter does not activate stat3 because it does not produce bft therefore ntbf does not contribute to colon cancer development but etbf does are tregs th17 and il17 good or badreduces intestinal il17 this process in a normal healthy condition tregs play an important role in inflammatory responses and immune homeostasis they express high levels of il2 receptor and produce endogenous il2 which inhibits the production of intestinal inflammation and prevents carcinogenesis however when etbf colonises a particular site of the colon it produces a large amount of bft damaging the intestinal mucosa to initiate etbftriggered colitis with the activation of the stat3 pathway this leads to direct contact between tregs and etbf and promotes tregs activation activated tregs lack the ability to produce endogenous il2 “ instead of producing endogenous il2 tregs consume exogenous il2 for their survival the consumption of exogenous il2 by tregs reduces the levels of exogenous il2 and produces an environment that favours the growth of th17 cells as the levels of il2 drop th17 cells are no longer inhibited and undergo expansion to produce a large quantity of naïve tcells this naïve subset of tcells then differentiates into th17 cells in excess wwwmjmsusmmy 0ccarcinogenesis this shows that colonisation of etbf promotes the accumulation of both tregs and th17 cells “ th17 cells start to produce large amounts of cytokines including tnf and il17 these cytokines promote cell survival and proliferation during injuries although th17 cells heal an injured site they turn into pathogenic th17 cells when deregulated these pathogenic th17 cells initiate chronic inflammatory condition il17 produced by pathogenic th17 cells are involved in an early inflammatory stage of the injuries it promotes tumour cell survival proliferation tumour neovascularisation and metastasis which allow “ additionally tumour cells and fibroblasts are stimulated by il17 to produce high amounts of angiogenic factors for angiogenesis il17 can activate stat3 pathway indirectly through il6 when il17 binds to il17 receptorbearing tumour cells it stimulates il6 production that is highly important for stat3 pathway activation as mentioned above this stat3 pathway activation contributes to several characteristics such as cancer proliferation antiapoptosis and angiogenesis that favour carcinogenesis in the colon this shows that there is a relationship between stat3 pathway and tregs in contributing to crc formation when etbf is accumulating in the intestinal tract as shown in figure to some extent stat5 and stat6 have been reported to be involved in inhibiting antitumour immunity when all stat3 and are activated together it highly enhances the tumourigenesis effect cleavage of ecadherin stimulate cell proliferationapart from inflammation bft alters the structure and function of colon epithelial cells by degrading ecadherin ecadherin is a 120kda glycoprotein that is the major structural protein in zonula adherens and is also known to be a tumour suppressor and zonula adherence protein this protein is responsible for the epithelial polarity in normal conditions the expression of ecadherin is linked to cellular functions including apoptosis and homotypic cell“cell “ unfortunately when ecadherin interacts with bft in the intestinal epithelial cells it degrades ecadherin rapidly in an atpindependent manner this cleavage promotes colonic injury inflammation and loss of membraneassociation resulting in morphological enhances cellular metastatic potential it is proven that changes and adhesion review article formation of colon canceretbfbftintestinal lumenepithelial cells†“ il2intestinal immune system†‘ th17tnfcell proliferation†‘ il17tregsinflammationcarcinogenesisil17 receptorbearing tumour cellsstat3 activationfigure the mechanism through abnormal systemof intestinal carcinogenesis immune factordependent the cleavage of ecadherin correlates with the changes of cell morphologies simultaneously degradation of ecadherin also promotes the binding of nuclear localisation of βcatenin and tcell transcriptional activator this binding promotes gene regulation and transcription additionally βcatenin plays an important role in wingless and int wnt signalling pathway promoting cell proliferation and epithelialmesenchymal transition and enhancing the expression of protooncogene in primary colorectal tumours cells in the centre of the tumour exhibit the presence of βcatenin and ecadherin however when the cells move away from the centre of the tumour they exhibit high amounts of nuclear βcatenin and the junction of ecadherin is lost important role ecadherin plays an in maintaining the morphology of cells there is a relationship with the ecadherin and the apical factin ring of the intestinal epithelial cells™ secretion when the loss of ecadherin increases the integrity of the apical factin is lost resulting in the increase in cell volume and chloride secretion and cell and epithelial barriers become wwwmjmsusmmy 0cmalays j med sci jul“aug “more permeable this contributes to intestinal inflammation diarrhoea and colon carcinogenesisalteration of the signalling pathway of colorectal cancerbft is involved in many colonic epithelial cell signal transductions when bft disturbs or activates the signalling pathway it brings adverse effects to the body and can lead to colorectal tumourigenesis figure the colonic epithelial cell signal transduction transpires through the nuclear factor kappalightchainenhancer of activated bcells nfκb wnt and mitogenactivated protein kinase mapk signalling pathways bft can stimulate nfκb pathway in the intestinal epithelial cells with the expression of heme oxygenase1 ho1 and cytokines to induce mucosal inflammation this pathway has the ability to enhance the survival of neoplastic cells by preventing them from undergoing apoptosis leading to tumour formation furthermore in figure it shows that when nfκb of intestinal epithelial cells is activated for a long time it induces the activity of nitric oxide synthase that breaks down larginine to produce nitric oxide which can damage cellular dna wnt signalling pathway is important to maintain the structures of the intestinal epithelium however wnt signalling pathway contributes negatively and affects cells which are extremely important for colorectal carcinogenesis and progression as wnt signalling pathway is activated it weakens tight junctions and reduces cellular adhesion this allows the cancer cells to undergo migration and metastasis hence cancerous cells can migrate to another ans spermine oxidase is a catabolic enzyme that increases ros which can be upregulated by bft in normal conditions figure ros acts as an important mediator in multiple cell signalling pathways and immune response that is produced naturally within biological systems it consists of superoxide hydroxyl radical and hydrogen peroxide however as the amount of ros becomes excessive it imparts negative effects in the disruption of redox homeostasis figure this excessive ros induces oxidative stress it oxidises cellular components including dna lipids and proteins within the cells etbfbftbftepithelial cellactivation of signaling pathwaynfκbwntnitric oxide synthase — reduce cell adhesion — weaken tight junction — cancer cell migratenitric oxidedamage cellular dnafigure the role of the signalling pathways when epithelial cells contact bftspermine oxidase smorelative oxygen species ros “ mediatorimmune responsemultiple cell signalling pathwaysfigure normal condition of the smo and ros that helps in immune response and cell signalling pathwayswwwmjmsusmmy 0conce the cellular components are oxidised it generates irreversible damage to host cells additionally ros plays an important role in the survival of cancer cells enhancing the effectiveness of carcinogenesis and aggravating cancer formed in the body “spermine oxidase smo “ upregulated†‘ relative oxygen species ros “ mediatordnalipidproteinirreversible damagecarcinogenesisfigure the adverse effect of smo contacted bftfamilial adenomatous polyposisfactors contributes the combination of both genetic and environmental to crc formation it is estimated that of crc development is due to genetic predisposition wherein nearly of all crcs are attributed to familial adenomatous polyposis fap fap is an autosomal dominant inherited disorder that describes the development of numerous colorectal adenomatous polyps these polyps are able to develop in the teenager™s colon meanwhile the number of polyps formed in the colon depends on the age of a person which means the number of polyps is directly proportional to the age of a person if these polyps are not removed from the colon they may transform from benign to malignant developing crc the source of fap disease is mainly due in the adenomatous to germline mutation polyposis coli apc gene “ this apc mutation occurs due to frameshifts insertions or deletions that may introduce a premature stop codon during the halfway through the transcription process these earlyintroduced premature stop codons in the gene sequence lead to incompletetruncated apc protein formation thus the normal function of apc protein is lost eventually facilitating carcinogenesis review article formation of colon canceradditionally germline mutations along with somatic mutations of the normal allele or loss of the normal allele lead to inactivation of apc once apc is inactivated it precisely commences carcinogenesis in normal conditions apc pathway acts as a gatekeeper controlling a part of wnt signalling pathway unfortunately when apc is mutated the function of apc pathway is lost or inactivated this inactivation of the apc pathway results in the activation of wnt signalling pathway this characteristic is mainly found in crc moreover apc mutation has the ability to alter bacteria“host epithelial interaction where it allows the bacteria to attach onto the mucosa if a person has the apcmutated gene and is exposed to etbf the chances of developing crc are high concurrently high amount of tumour load is displayed in the person™s colon conclusionthe human gastrointestinal tract contains its own bacterial flora that benefit humans daily b fragilis is one of them and consists of two classes namely ntbf and etbf the differences between both the classes is the presence of bft etbf is able to produce bft that can disrupt the intestinal environment and promotes inflammation simultaneously bft degrade ecadherin and causes inflammation ibd is a chronic intestinal inflammation associated with etbf and can induce crc however patients with cd have lower risk of developing crc as compared to those with uc patients with ibd exhibit stat3 activation due to the stimulation of immune response that favours th17 cell generation as the levels of th17 cell increase it brings a huge disadvantage to the intestinal tract due to the production of il17 furthermore il17 stimulates the production of il6 that is required to activate stat3 this indicates that the stat3 pathway activates for a long time longterm stat3 activation blocks antitumour immune response which supports the growth of cancer cells thus stat3 th17 and il17 are highly important in carcinogenesis concurrently the production of proinflammatory cytokines at the site of inflammation triggers the production of cox2 enzymes that release pge2 cox2 is also known for its carcinogenic abilities due to the production of pge2 that controls cell proliferation additionally bft affects signal transductions such as wnt wwwmjmsusmmy 0cmalays j med sci jul“aug “nfκb and mapk signalling pathways and induces tumourigenesis considering that bft induces inflammation activates stat3 and alters signalling pathways it can be concluded that bft produced by etbf plays an important role in colon carcinogenesis boleij a hechenbleikner em goodwin ac badani r stein em lazarev mg et al the bacteroides fragilis toxin gene is prevalent the colon mucosa of colorectal cancer in patients clin infect dis “ httpsdoi101093cidciu787acknowledgementsnoneconflict of interestnonefundsnoneauthors™ contributionsconception and design hkkdrafting of the article with supervision cwtcritical revision of the article for important intellectual content fdfinal approval of the article hkk fdcorrespondencedr fabian davamaniphd microbiology university of madrasfaculty of biomedical scienceschool of health sciences international medical university kuala lumpur malaysia tel fax email fabian_davamaniimuedumyreferences snezhkina av krasnov gs lipatova av sadritdinova af kardymon ol fedorova ms et al the dysregulation of polyamine metabolism in colorectal cancer is associated with overexpression of cmyc and cebpβ rather than enterotoxigenic bacteroides fragilis infection oxid med cell longev “ httpsdoi10115520162353560 sears cl geis al housseau f bacteroides fragilis from carcinogenesis j clin symbiont invest “ httpsdoi101172jci72334subverts mucosal to biology colon lv y ye t wang h zhao j chen w wang x et al suppression of colorectal tumorigenesis by recombinant bacteroides fragilis enterotoxin2 in vivo world j gastroenterol “ httpsdoi103748wjgv23i4603 pierce jv bernstein hd genomic diversity of enterotoxigenic strains of bacteroides fragilis plos one 2016116e0158171 httpsdoi 101371journalpone0158171 sun j kato i gut microbiota inflammation and colorectal cancer genes dis “ httpsdoi101016jgendis201603004 erdrich j zhang x giovannucci e willett w proportion of colon cancer attributable to lifestyle in a cohort of us women cancer causes control “ httpsdoi101007s105520150619z mughinigras l schaapveld m kramers j mooij s neefjesborst ea van pelt w et al increased colon cancer risk after severe salmonella infection plos one 2018131e0189721 httpsdoi101371journalpone0189721 francescone r hou v grivennikov si microbiome inflammation and cancer cancer j “ httpsdoi101097ppo0000000000000048 wick ec rabizadeh s albesiano e wu x wu s chan j et al stat3 activation in murine enterotoxigenic bacteroides inflamm bowel dis “ httpsdoi101097mib0000000000000019induced by fragili
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"colorectal cancer crc remains the third most prevalent cancer type and leading cause of cancerrelated deaths with million cases and deaths worldwide during the occurrence and progression of crc result from a wide array of cellular transformation processes which include genetic and epigenetic mutations that drive uncontrolled cell proliferation and escape from apoptosis2“ chemotherapy and surgery remain the major therapeutic treatment for crc patients5 fluoropyrimidinebased chemotherapy eg 5fluorouracil has been used as the firstline systemic chemotherapy of treating advanced crc for over a half century6 however most patients receiving chemotherapy finally develop drug resistance which is considered to be the major reason for crc therapy failure7 furthermore even though chemotherapy has significant antitumor activity the side effects can affect the quality of a patient's life which makes the new therapeutic approaches urgentdrug design development and therapy “ sun 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 0csun dovepresstraditional chinese medicines such as dendrobium have been shown to exert anticancer activity in many kinds of cancers89 erianin 2methoxy5[2345trimethoxy phenylethyl]phenol figure 1a a natural compound derived from dendrobium candidum shows various pharmacological activities and therapeutic potential to inhibit multiple cancers in vivo and in vitro10“ li demonstrated that erianin inhibited the proliferation of acute promyelocytic leukemia hl60 cells by regulating the expression of bcl2 and bax10 in addition erianin caused moderate growth delay in xenografted human hepatoma bel7402 and melanoma a37511 furthermore erianin induced cell cycle g2mphase arrest and apoptosis via the jnk signalling pathway in osteosarcoma and bladder cancer1213 erianin can also inhibit cell invasion metastasis and angiogenesis in lung cancer and breast cancer by the figure erianin inhibited crc cells growth a chemical structure of erianin b and c sw480 and hct116 cells were treated with indicated concentration b and time c of erianin cell viability was assessed by cck8 assay p ˂ p ˂ d and e ncm460 cells were treated with indicated concentration d and time e of erianin cell viability was assessed by cck8 assay f sw480 and hct116 cells were performed colony formation assay after being treated with indicated concentration of erianinsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun regulation of ido mpp and timp expressions1415 interestingly besides the function on cell growth apoptosis and migration erianin was found to strongly affect the serum levels of cytokines and immune response in liver cancer16 more importantly in addition to the anticancer effects previous a study also suggested that erianin had no major anrelated toxicities12however to the best of our knowledge neither the mechanism nor the effect of erianin on colorectal cancer has been reported hence in this study we evaluate the antitumor potential and molecular mechanisms of erianin in human colorectal cancer sw480 and hct116 cells and provide a theoretical basis of erianin application for colorectal cancer therapymaterials and methodsmaterialsantibodies against cleaved parp cat bak cat bax cat bcl2 cat bclxl cat catenin cat cyclin d1 cat cmyc cat hdac2 cat and gapdh cat were purchased from cell signaling technologies danvers ma usa antibody against αtubulin cat t6199 was purchased from sigma aldrich co st louis mo usaerianin was purchased from shanghai yuanye bio technology co ltd china and dissolved in dmso wntcatenin signaling inhibitor wnt974 was purchased from medchemexpress monmouth junction nj usa and dissolved in dmsocell culturethe human colorectal cancer cell lines sw480 and hct116 were purchased from american type culture collection atcc manassas va usa cells were maintained in rpmi1640 medium supplemented with fbs thermo fisher scientific waltham ma usa uml penicillin and µgml streptomycin thermo fisher scientific and cells were cultured at °c with co2cell viability and colony formation assaycell viability was assessed with the cell counting kit cck8 dojindo japan according to the manufactorer™s instructionsfor the colony formation assay crc cells cells well were seeded in a sixwell plate and maintained in medium for “ days subsequently the colonies were fixed with paraformaldehyde and stained with crystal violet and the number of clones was counted using an inverted microscopekit quantitative realtime pcr qrtpcrtotal rna from crc cells was isolated using rna isolation kit omega norcross ga usa according to the manufacturer™s protocol total rna µg was used as the template for cdna synthesis by using iscripttm reverse transcription super mix biorad laboratories inc hercules ca usa before the samples were analyzed using sybr green master mix on a realtime pcr system biorad laboratories inc the primer sequences used were as follows cmyc forward 5ʹ aaacacaaacttgaaca gctac3ʹ reverse 5ʹ atttgaggcagtttacatt atgg3ʹ cyclin d1 forward 5ʹaggcggatgagaac aagcaga3ʹ reverse 5ʹcaggcttgactccagaag gg3ʹ cd47 forward 5ʹggcaatgacgaaggaggt ta3ʹ reverse 5ʹatccggtggtatggatgaga3ʹ and gapdh forward 5ʹcacccactcctccacctttg3ʹ and reverse 5ʹccaccaccctgttgctgtag3ʹ the 2δδcq method was used to calculate the relative expression levelswestern blottingfor western blotting μg cellular protein extracts were separated in sdspage gel and were then transferred to nitrocellulose membranes emd millipore burlington ma usa the membrane was blocked with nonfat milk and incubated with primary antibodies overnight at ° then the membranes were incubated with secondary antibody and the proteins were visualized using super signal west pico chemiluminescent substrate thermo fisher scientifictransit transfectionplasmid pegfpn1betacatenin was purchased from addgene watertown ma usa lipofectamine thermo fisher scientific carlsbad ca usa was used for transit transfection according to the instructionscatenin sirna was purchased from sigmaaldrich co lipofectamine rnaimax thermo fisher scientific was used for transfection according to the instructiondrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepresscell cycle analysisafter treated with vehicle or indicated drugs crc cells were harvested by trypsinization fixed with ethanol and retained at ˆ’°c overnight after cells were centrifuged and washed with pbs they were resuspended in propidium iodide pi solution containing rnase μgml in the dark at room temperature for min and then studied in a flow cytometercaspase37 activity assayapoone„¢ homogeneous caspase37 assay promega corporation madison wi usa was used to measure caspase37 activity briefly apoone® homogeneous caspase37 reagent μlwell was added to a 96well plate and the plate was then placed on a shaker for five minutes “ rpm before incubating for h at room temperature the reading of each well was measured by spectrofluorometerapoptosis assay by annexin vannexin vfitc staining was used to detect the extent of apoptosis induced by erianin briefly crc cells were treated with erianin for h and were then collected and resuspended in μl annexin vbinding buffer and μl pi for minat room temperature in the dark then the cells were finally analyzed by the flow cytometry bd facs calibur with an emission filter of nm for pi red and “ nm for fitc greenapoptosis assay by dapithe effect of erianin on apoptosis induction was evaluated by dapi staining assay crc cells × were seeded in a 96well plate after treatment the cells were washed three times with pbs and paraformaldehyde was added to each well for fixation after permeabilization with triton x100 solution dapi solution was added the cells with condensed and fragmented chromatin were analyzed by echo fluorescence microscopycellular thermal shift assayfor cellular thermal shift assay crc cells were pretreated with μm mg132 for one hour and then incubated with erianin for four hours after washing with icecold pbs cells were aliquot into pcr tubes μl each and incubated at different temperatures for four minutes after being frozen and thawed twice using liquid nitrogen cells were centrifuged and proteins were analyzed by western blottingtopfop luciferase reporter assaythe transcriptional activity of catenin was assessed using the topfop dualluciferase reporter system dual glo„¢ luciferase assay system promega the renilla luciferase plasmid prltk promega which controls for transfection efficiency was cotransfected with catenin responsive firefly luciferase reporter plasmid topflash emd millipore or the negative control fopflash emd millipore using the lipofectamine thermo fisher scientific cells were harvested after h in culture and the luciferase activity was determined by the luciferase assay system promega using a microplate luminometer berthold bad wildbad germanyflow cytometry analysiserianin treated crc cells were washed and resuspended in μl facs buffer and stained with fitcconjugated anticd47 bd biosciences san jose ca usa antibodies all samples were incubated for minutes at °c and then washed twice with facs buffer flow cytometry analyses were performed on bd facs canto iiin vitro phagocytosis assayfor phagocytosis assay thp1 derived macrophages were seeded in a sixwell tissue culture plate erianintreated crc cells were washed and labeled with μm of carboxyfluorescein succinimidyl ester cfse thermo fisher scientific after incubating macrophages in serum free medium for two hours cfselabeled crc cells were added to the macrophages for another two hours at °c macrophages were then washed and imaged with an inverted microscope the phagocytosis efficiency was calculated as the number of macrophages containing cfse labeled crc cells per macrophageschromatin immunoprecipitation chip assaychip assays were performed using the simplechip® enzymatic chromatin ip kit cell signaling technologies according to manufacturer's instructions using the antibodies against h3k27ac immunoprecipitated dna was analyzed by qrtpcr using the following primers cd47 promoter fragment f ²aggatgaatgatgtggcctgt3² and r ² caaacaggcattagcagcgt3² fragment f submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun ²ggggatgtgttggatacgct3² and r ² ctctg cgttcggctcgtcta3² fragment f ²agggaag agcagagcgagta3² and r ² ttgctttcactcc caccctc3² fragment f ²agagagaggacag tggggc3² and r ² ccagtcgcaggctccaga3² fragment f ² gccgcgtcaacagca3² and r ² aaaggcatcattcttggaaattgt3²with ¨° sw480 cells per mouse suspended in vivo xenograftnodscid shanghai slac laboratory animal co ltd china mice were injected subcutaneously in right flank in µl pbs and mixed with an equal volume of matrigel animals with tumors volume mm3 were divided into two groups n6 and treated with either placebo or mgkg erianin for continuously three weeks by intraperitoneal injection tumor size were measured at the indicated times all the animalrelated procedures were approved by the animal care and use committee of the changchun university of chinese medicine all animal experiments were conducted according to the nih guide for the care and use of laboratory animalsstatistical analysisdata were presented as mean ±sd from three independent experiments p value was determined using paired student™s ttest and a p value ˂ was deemed to indicate statistical significanceresultserianin inhibited crc cell growthfigure 1a illustrates the chemical structure of erianin to investigate the inhibitory effect of erianin on crc cell viability we treated two crc cell lines sw480 and hct116 with different concentrations of erianin and nm for and h as shown in figure 1b and c erianin treatment significantly inhibited the viability of crc cells in a dose and timedependent manner importantly erianin did not show cytotoxic effects on normal human colon mucosal epithelial cell line ncm460 figure 1d and e in addition consistent with the shortterm growth assay our colony forming unit assay also showed that erianin inhibited the colony formation ability of sw480 and hct116 cells figure 1ferianin elevated cell cycle arrest and apoptosisto verify the causal relation of cell viability inhibition the cell cycle distribution was analyzed erianin increased cell number at g2m phase but decreased cell number at s and g0g1 phases after 24h incubation with indicated concentration in sw480 and hct116 cells figure 2a and b to explore the effect of erianin on apoptosis we examined the activity of caspase the protein level of cleaved parp bax bak bcl and bclxl as shown in figure 2c“e the activity of caspase protein level of cleaved parp bak and bax pro apoptosis increased as the concentration of erianin increased in contrast the protein level of bcl2 and bclxl anti apoptotic decreased after erianin treatment figure 2e annexin v flow cytometry and dapi staining further confirmed that erianin could induce cell apoptosis figure 2f and gerianin inhibited catenin translocationincreasing evidence revealed that the wntcatenin pathway plays critical role in colorectal cancer tumorigenesis we hypothesized that erianin might have effect in modulating the wntcatenin pathway first we investigated the effect of erianin on catenin phosphorylation as shown in figure 3a no obvious change was observed on catenin phosphorylation level we then evaluated the effect of erianin on catenin translocation as shown in figure 3b“e catenin expression in cytoplasm was increased whereas expression in the nucleus was decreased with the treatment of erianin in a dose and timedependent manner to further explore the effect of erianin on catenin transcription activity we performed topfop dual luciferase assay we found that topfop relative luciferase activity was significantly decreased after erianin treatment both in sw480 and hct116 cells figure 3f and gerianin bound catenin directlysince erianin inhibited catenin translocation to the nuclear without changing its phosphorylation level we hypothesized that erianin might bind catenin directly to determine whether erianin physically binds catenin we performed a cellular thermal shift assay the results from this experiment indicated that erianin treatment increased the thermal stability of catenin when cells were pretreated with the proteasome inhibitor mg132 for one hour figure 4a and b in contrast erianin treatment had no effect on the thermal stability of gapdh a loading control figure 4a and b these results strongly suggested a specific physical interaction between erianin and catenindrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin elevated cell cycle arrest and apoptosis a and b sw480 and hct116 cells were treated with erianin for h and then analyzed by pi staining to determine cell cycle phase distribution c sw480 and hct116 cells were treated with erianin for h the relative caspase37 activity was measured using apoone„¢ homogenous caspase37 assay p ˂ p ˂ d and e the protein level of cleaved parp1 bak bax bcl2 and bclxl were analyzed by western blotting after treated with indicated concentration of erianin f and g sw480 and hct116 cells were treated with erianin for h apoptosis was assessed using annexinv flow cytometry analysis f or dapi staining gsubmit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited catenin translocation a the protein level of indicated proteins was analyzed by western blotting after being treated with indicated concentration of erianin for h b“e the protein level of catenin in cytosol and nucleus was analyzed by western blotting after treated with erianin for indicated concentration b and c and time d and e f and g sw480 and hct116 cells were treated with erianin for indicated concentration f and time g the transcriptional activity of catenin was assessed by topfop luciferase reporter assay p ˂ p ˂erianin inhibited the expression of cmyc and cyclin d1as cmyc and cyclin d1 are the direct targets of the wnt catenin pathway we then evaluated the mrna and protein level of cmyc and cyclin d1 unsurprisingly both mrna and protein level of these two proteins were significantly decreased after erianin figure 5a“c interestingly no synergetic effect was observed when combining erianin with wntcatenin signaling inhibitor wnt974 which indicated that erianin regulates cmyc treatment drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin interacted with catenin a and b sw480 a and hct116 b cells were treated with μm mg132 for one hour followed by four hours incubation with nm erianin before performing thermal shift assay the lower panel shows the charts of percentages of nondenatured protein fractionand cyclin d1 via wntcatenin signaling figure 5d furthermore the inhibitory effect of erianin on cmyc and cyclin d1 expression and cell viability could be reversed by catenin overexpression figure 5e and f which indicated that erianin regulates crc cell growth via catenincd47 mediated phagocytosis we used an in vitro assay by coculturing thp1 derived macrophage with crc cell lines sw480 or hct116 as shown in figure 6g and h treatment of erianin markedly promote colorectal cancer cell phagocytosis by macrophages these results suggest that erianin treatment can attenuate cd47 expression and ultimately promote phagocytosis of crc cellserianin decreased cd47 expression and increased phagocytosisthe immune checkpoint protein cd47 is included in the list of wntcatenin target molecules with a role in immunity escape17 since catenin depletion by sirna inhibited the expression of cd47 figure 6a we then sought to know whether erianin regulates the expression of cd47 first we explored the effects of erianin on cd47 mrna protein and cell surface level in both sw480 and hct116 cells erianin treatment significantly decreased the mrna protein and cell surface level of cd47 figure 6b“d promoter analysis by ucsc genome browser demonstrates that h3k27 acetyl marks are enriched in cd47 promoter regions figure 6e next our chip assay demonstrated that h3k27ac enrichment specifically near promoter region f3f5 was significantly decreased with erianin treatment figure 6f to investigate the effect of erianin on erianin inhibited tumor growth in vivoto investigate the possibility of erianin as a potential therapy in crc we tested the function of erianin on tumor growth in a mouse model the mouse model was established by s c injection of sw480 cells into nodscid mice after three weeks treatment we analyzed the tumor size and weight as shown in figure 7a“c the tumor size and weight from the erianin treatment group were significantly lower than that from the control group in addition after days of bearing tumor the weight of the mice had no significant change figure 7dto examine the impact of therapy on catenin and its downstream signaling localization of catenin protein level of cd47 cmyc bcl2 and bax three representative tumors from each group were analyzed using western blotting as shown in figure 7e and f catenin expression in cytoplasm was increased whereas expression in nucleus was decreased with the treatment of erianin the submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited the expression of cmyc and cyclin d1 a“c after treated with indicated concentration and time of erianin mrna and protein level of cmyc and cyclin d1 were analyzed by qrtpcr and western blotting p ˂ d sw480 cells were treated with erianin orand wnt974 for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and f sw480 cells were treated with erianin for h followed by overexpression with catenin plasmid for h protein levels of cmyc and cyclin d1 were analyzed by western blotting e and cell viability was assessed by cck8 assay f p ˂protein level of cd47 cmyc and bcl2 decreased while bax increased after erianin treatment these data indicated that erianin inhibited tumor growth via catenin in vivodiscussioncrc is one of the most malignant and commonly diagnosed solid tumors all around the world18“ although crc incidence rates have declined somewhat chemotherapies are inefficient in most crc patients due to resistance2122 thus the development of acquired therapeutic drugs researching novel and safe treatment strategies is essential for improving the prognosis of crc patients in recent years natural medicinal plants are receiving more and more attention and considered to be important sources of treatment23 novel dendrobium is considered as one of the most important herbs in the orchidaceae family and shows diverse pharmacological functions including anticancer neuroprotective antidiabetic and immunemodulating activities24 erianin derived from dendrobium is one of the most for cancer drug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressfigure erianin decreased cd47 expression and increased phagocytosis a sw480 cells were transfected with nontarget nt or catenin sirna for h protein levels of indicated protein weres measured by western blotting b“d sw480 and hct116 cells were treated with erianin for indicated dose the mrna level b protein level c and cell surface cd47 d were detected by qrtpcr and flow e the ucsc genome browser revealed the enrichment of h3k27ac on cd47 promoter f the enrichment of h3k27ac on cd47 promoter f1f6 was detected by chip assay g and h sw480 and hct116 were treated with indicated concentration of erianin for h representative images showed the effect of erianin on phagocytosis g and bar graphs showed quantitative analysis of phagocytosis h p ˂ p ˂submit your manuscript wwwdovepresscom dovepress drug design development and therapy 0cdovepress sun figure erianin inhibited tumor growth in vivo a typical photos of tumors from the control and erianin treated groups b and c erianin decreased tumor volume and weight p ˂ d mice body weight of control and erianin treated groups was measure at indicated time e the protein level of catenin in cytosol and nucleus in three representative tumors from mouse to mouse of each group were analyzed by western blotting f the protein level of indicated protein in three representative tumors from mouse to mouse of each group were analyzed by western blottingdrug design development and therapy submit your manuscript wwwdovepresscom dovepress 0csun dovepressnoteworthy constituents that have been used as an antipyretic and an analgesic in traditional chinese medicine25 recently several studies have proved that erianin shows significant antitumour activity in a variety of human cancer cells10ˆ’ consistent with literature in this study we found that erianin had a significant antiproliferative effect against crc cells the inhibitory effect caused by erianin may result from induction of apoptosis and arrest of cell cycle at g2m since the effect of erianin on crc cells has never been studied before we further confirm its antitumor activity in a mouse model which indicated that erianin significantly inhibited tumor growth in vivoseveral signaling pathways including egfrmapk pi3kakt or wntcatenin have been linked to crc genesis and progression26 as the aberrant activation is present in almost all crc cases wntcatenin signaling is prominent among these pathways27 inactivated mutations in the apc gene leads to stabilization and ensuing nuclear translocation of catenin to facilitate tcflef dependent transcription of wntcatenin signaling target genes such as cmyc and cyclin d1 to drive cell proliferation survival and metastasis28“ to understand the mechanisms of action of erianin we assessed the effect of erianin on wntcatenin pathway interestingly we found that erianin treatment had no effect on catenin phosphorylation but inhibited the translocation of catenin in the nucleus which suggested to us that erianin physically interacts with catenin our cellular thermal shift assay confirmed this hypothesis the thermal stability of catenin increased after erianin treatment as catenin downstream targets the expression level of cmyc and cyclin d1 significantly decreased after erianin treatmentcd47 a transmembrane glycoprotein expresses ubiquitously and mediates a œselfdonoteatme signal on normal cells however cd47 is often upregulated in tumor cells to evade innate immunity31“ anticd47 antibodies which block cd47 sirpα interactions and promote macrophage mediated phagocytosis of tumor cells has shown promise in several solid tumors31 in colorectal cancer cd47 promotes colon cancer cell migration and metastasis34 in addition upregulated immuneescape pathways such as cd47 sirpα are responsible for immune escape and survival in circulating tumor cells of colorectal cancer35 myc an oncogene identified as a wntcatenin target gene was reported to control cd47 transcription therefore mutations in components of the wntcatenin signaling pathway which induced
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"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 conflictingstudies 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 identified 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 œangiogenicswitch of tumor cells is turned on during tumor progression the œangiogenic 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 firstly 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 fenofibrate a clinically used ppar alpha agonist moreover fenofibrate 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 fibroblast 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 fibrosarcoma 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 thespecific 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 anti‚ammatory 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 first 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 anti‚ammatory 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 firstlysuggesting the promotion for angiogenesis upon pparbetadelta activationmüllerbrüsselbach show that ppar betadelta mice implanted with llc and b16 melanoma exhibit diminished blood flow 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 musclespecific ppar betadelta overexpression leads toppar betadeltaan increase in the number of oxidative muscle fibers 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 specificity 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 significantly 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 anti‚ammatory 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 betadeltaspecificsirna 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 conflicting studies indicating that the functional role of ppar betadelta in tumorigenesis or carcinogenesis still remains highly controversial [“] and dependent on specific 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 œhubnode transcriptional factor which governs a tumor œangiogenic 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 tumorltrating 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 intumorltrating 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 deficiencyin 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 fibrotic 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 endotheliumspecific ppar betadelta overexpression in vivo leads to 0cppar researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ecspecificppar betadelta action mechanism in tumor progressionindependent of some controversial observations of pparbetadelta in specific tumor or cancer cell types wagner also firstly 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 mmp9pro‚ammatory mediators such as cox2 and cytokinesand chemokines such as il1 and cxcl8 even some ofwhich have been further identified 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 pro‚ammatorymediator 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 œangiogenic 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 beenidentified 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 tumorltrating ‚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 significantly 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 pro‚ammatory 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 significantly 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 pro‚ammatory mediators chemokines and proangiogenic factors closely communicate with 0cppar researcheach other and also associate with tumorltrating 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 cancerassociatedfibroblasts 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 specificecderived 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 significantly upregulated in the vascularppar betadelta overexpression model in vivo andwt1 is also identified 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 identified 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 stromalfibroblasts may mediate pdgfbinduced tam recruitment thus implicating a role of pdgfr beta in tumorstroma to facilitate tumor progression most recently it wasfurther shown that specific 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 identifiedas 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 identified 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 œhallmark 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 tumorltrating ‚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
the heterogeneity of cancer cells is generally accepted and astem celllike subpopulation that is called œcancer stem cellscscs has been identified in various types of malignanttumors although the lack of consensus on the definitioncscs are widely recognized as a small subpopulation amongcancer cells with the properties of selfrenewal and tumor initiation as cscs play a critical role in the recurrence andmetastasis of cancer targeting the cscs is thought to bea promising approach for curing cancera large number of past studies have tried to identify andcharacterize the cscs as normal tissuespecific stem cellsare considered as the main origin of cancer the cscsare also thought to be inherited at least partially the characterization of normal tissuespecific stem cells thereforemany studies on the identificationpurification of cscs havesimply shared markers of hematopoietic stem cells includingthe most popularly used cell surface markers of cd44 andcd133 [ ] cd44 is a type i transmembrane glycoproteinthat is expressed on hematopoietic fibroblastic and glial cellsand functionally known to mediate cellcell and cellmatrixinteractions previous studies have demonstrated that thecd44 is not only a biomarker but also plays critical roles inthe maintenance of cscs the resistance to various therapiesstresses and the metastasis of cancer cells [“]cd133 is originally identified as protein expressing on thecell surface of hematopoietic stem cells and has subsequently been found to be critical in the maintenance ofœstemness of stem cells in various tissues [“] cd133has also been found in some csc [“] which contributesto therapeutic resistance through the activation of akt bcl2and mapkpi3k signaling pathways [“] although theexpressions of cd44 and cd133 in cancer cells likely associate with the resistances to radiotherapy chemotherapy andvarious stresses the diï¬erent significance between cd44and cd133 has not yet been well understoodin this study we investigated whether the expression ofcd44 and cd133 in human colorectal cancer cells hct8diï¬erently contributed to drug resistance our data indicated 0cstem cells internationalthat the expression of cd133 rather than cd44 closely associated with doxorubicin dxr resistance at least partiallythrough drug excretion and redox regulation materials and methods cell culture human colorectal cancer hct8 cells werecultured in rpmi medium fujifilm wako purechemical japan supplemented with fbs gibco°thermo fisher scientific ma usa at c in a humidifiedatmosphere of air and co2 separation of cd44 and cd133positive cells fromhct8 cells we separated the parent hct8 cells intocd44positive cd44 and cd133positive cd133 cellsby a twostep magnetic cell sorting method as described previously [ ] briefly hct8 cells were collected as a singlecell suspension by trypsinization and then incubated withmagnetic microbeadconjugated antihuman cd44 antibodymiltenyi biotec germany for min after washing cellswere separated into cd44 and cd44 subpopulations byusing the automacs„¢ pro separator miltenyi biotecaccording to the manufacturer™s instruction the purifiedcd44 cells were further expanded and then harvested as asinglecellsuspension to be incubated with magneticmicrobeadconjugated antihuman cd133 antibody miltenyibiotec for min after washing the cd44cd133 andcd44cd133 subpopulations were separated as describedabove this twostep isolation enabled us to obtain a sufficientnumber of cd44 cd44 cd44cd133 and cd44cd133 cells for our experimentsto verify the purity of each subpopulation isolated cellswere stained with pelabelled mouse antihuman cd133clone ac133 miltenyi biotec and fitclabelled mouseantihuman cd44 clone db105 miltenyi biotec according to the supplied protocols flow cytometry analysis wasperformed using a facscalibur becton dickinson asdescribed previously mouse igg1pe miltenyi biotecand mouse igg1fitc miltenyi biotec were used as a negative control cytotoxicity assays cells were seeded in 96well cultureplates at a density of — cells per well and cultured overnight the cells were then treated with various concentrationsof dxr fujifilm wako pure chemical in the absence orpresence of verapamil fujifilm wako pure chemicalcytotoxicity assays were performed using the cell proliferation kit i mtt roche applied science germany asdescribed previously the absorbance was measured at nm using a microplate reader multiskan fc thermofisher scientific analysis on the expression of abc transporters theexpressions of the atpbinding cassette subfamilies of bmember abcb1 or g member abcg2 were analyzedby flow cytometry briefly cells were incubated with mouseprimary antibodies against human abcb1 and abcg2bd biosciences ca usa and then labeled by fitcconjugated antimouse igg bd biosciences according tothe manufacturer™s instruction respective isotype controlswere used as a negative control after washing flow cytometry analysis was performed using a facscalibur analysis of cellular accumulation of dxr the intracellular accumulation of dxr was analyzed by flow cytometrybriefly cells were treated by μm dxr for hr in theabsence or presence of μm verapamil or μm buthionine sulfoximine bso sigmaaldrich mo usa cellswere then collected as a singlecell suspension and washedtwice with icecold phosphatebuï¬ered saline the accumulation of dxr within cells was evaluated by the intracellularfluorescence intensity using a facscalibur the nucleusaccumulation of dxr was analyzed by using cell pelletstreated with triton x100pbs as assay material asdescribed previously expressionlevelsanalysisimmunoblot detection of intracellular ros the intracellular roslevel based on the oxidation of ²²dichlorodihydrofluorescein diacetate h2dcfda molecular probes thermofisher scientific was measured to form the fluorescent compound ²²dichlorofluorescein dcf using a facscaliburofphosphorylatedp38 map kinase phosphop38mapk totalp38mapk and nuclear factor erythroid 2related factor nrf2 in the cells were estimated by immunoblotting brieflycell lysate μg of total protein was separated by sodiumdodecyl sulfate“polyacrylamide gel electrophoresis sdspage gel transferred to pvdf membranes biorad causa and then incubated with primary antibodies cell signaling technology ma usafollowed by appropriatehrplabeled secondary antibodies dako agilent pathologysolutions ca usa blots were developed by enhancedchemiluminescence using an ecl kit ge healthcare lifesciences pa usa semiquantitation was done by measuringthe density of bands using the image quant las minibiomolecular imager ge healthcare life sciences asdescribed previously sirna treatment smallinterfering rna sirnaspecific targeting to cd133 on targetplus sirnaand a scramble sirna on targetplus sirna negativecontrol were obtained from dharmacon horizon discovery cambridge uk cells were seeded in 6well plates — cellswell and incubated for hr transfectionswere performed using dharmafect sirna transfectionreagents dharmacon according to the manufacturer™sinstructions analyses were done at hr after sirnatransfection statistical analysis all of the results are presented as themeans ± sd statistical significance was determined by oneway analysis of variance anova followed by tukey™s testdr spss ii chicago il diï¬erences were considered significant when p results hct8 cells were separated into various subpopulationsbased on their expressions of cd44 and cd133 first we 0cstem cells internationalparent cellshlffl1hdccd44hlfhlfcd44ˆ’fl1hcd44fl1hahlfhlfcd44cd133ˆ’fl1hcd44cd133fl1hparent cd44ˆ’cd44 cd44133ˆ’ cd44133enilesab fo noitarefilorp llecparent cd44ˆ’ cd44 cd44ˆ’cd44bcfigure continued 0cstem cells internationalenilesabstnuo0ccd44cd133ˆ’ ± stnuo0ccd44cd133 ± fl2hfl2hsyad stnuo0c ± fl2hstnuo0cd ± fl2hfigure the separation of hct8 colorectal cancer cells into diï¬erent subpopulations based on the expression of cd44 and cd133 arepresentative dot plots of flow cytometry analysis show the purities of each subpopulation of isolated cells quantitative data in the dotplots are presented as the percentages of positive cells from three independent experiments b representative photos of morphologicalproperties upper and mtt assay on cell growth lower at hr after the initiation of culture data are presented as the mean ± sd fromthree independent experiments c d representative histograms of flow cytometry analysis showed the expressions of cd44 c andcd133 d at baseline and days after cell culture the dotted vertical lines through histograms indicate the diï¬erence in the expressionpeaks between the baseline and at days after culture quantitative data in the histograms are presented as the mean fluorescentintensity from three independent experimentsseparated the hct8 cells into cd44 and cd44 subpopulations and compared their sensitivity to anticancer drugs ofdxr and cisplatin cisdiaminedichloroplatine cddphowever no diï¬erence in the sensitivity to the two drugswas observed between cd44 and cd44 cells data notshown we further tried to purify a small population ofcd133 cells from these cd44 cells cd44 cells almostnegatively expressed with cd133 figure 1a as a resultwe separated hct cells into diï¬erent subpopulationsincluding cd44 cd44 cd44cd133 and cd44cd133 cells the purities of isolated cells in each subpopulation were confirmed to be around by flow cytometryfigure 1aandphenotype changein diï¬erent growthsubpopulations of cells the morphology and proliferationof these cells could not be found obviously diï¬erent amongsubpopulations figure 1b the expression of cd44 in allsubpopulations kept stable within days of reculturingfrom the frozen cells that stocked immediately after isolationinterestingly the expression of cd44 was a tendency todecrease with culture time in cd44 fluorescence intensity ± at baseline vs ± at days p figure 1c and cd44cd133 cells fluorescence intensity ± at baseline vs ± at days p figure 1c but still kept stable in cd44cd133 cells at days after reculturing fluorescence intensity ± at baseline vs ± at days p figure 1cthe expression of cd133 in cd44cd133 cells kept verystable fluorescence intensity ± at baseline vs ± at days p figure 1d and cd44cd133cells did not turn to express cd133 within days of reculturing fluorescence intensity ± at baseline vs ± at days p figure 1d therefore we usedthe cells within days after reculturing from the frozenstocked cells in subsequent experiments dxr resistance of cd44cd133 cells next we evaluated the sensitivity of cells to dxr by mtt assay withthe addition of μm of dxr in medium we foundthat the survival of cd44cd133 cells was significantlyhigher than all other subpopulations of cells after hr of 0cstem cells international ytilibaiv llecŽŽŽŽŽðœ‡mparent cellscd44ˆ’ cellscd44 cellscd44133ˆ’ cellscd44133 cellsdxrbso003hct8doxb24 hfl3h006hct8 cd44ˆ’doxb24 hfl3h009hct8 cd44doxb24 hsllec tnerapstnuocsllec ˆ’dcstnuocsllec dcstnuocdxr001hct8dox24 hfl3hstnuocadxrverapamil002hct8doxv24 hstnuocfl3h004hct8 cd44ˆ’dox24 h005hct8 cd44ˆ’doxv24 hstnuocstnuocfl3hfl3h007hct8 cd44dox24 h006hct8 cd44doxv24 hstnuocstnuocfl3hfl3hsllec ˆ’dcsllec dc006hct8 cd44cd133ˆ’dox24 h006hct8 cd44cd133ˆ’doxv24 hstnuocstnuocstnuocfl3hfl3h00hct8 cd44cd133dox24 h00hct8 cd44cd133doxv24 hstnuocfl3hstnuocstnuocfl3hbfigure continuedfl3h012hct8 cd133ˆ’doxb24 hfl3h015hct8 cd44cd133doxb24 hfl3h 0cstnuocstem cells internationalabcg2abcb1stnuocfl1hparent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cells ± ± ± ± ± parent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cellscfl1h ± ± ± ± ± figure dxr resistance of diï¬erent subpopulations of cells a mtt assay was done to evaluate the cytotoxicity of dxr data are expressedas the percentile of baseline before dxr treatment from three independent experiments ˆ—p vs all other subpopulations brepresentative histograms of flow cytometry analysis show the accumulation of dxr in cells hr after the treatment with μm dxrin the absence or presence of μm verapamil and μm bso the dotted vertical lines through histograms indicated the mean levels ofdxr accumulation in cd44cd133 cells for comparing with other subpopulations of cells the results were reproducible in threeindependent experiments c representative histograms of flow cytometry analysis show the expression of the abcb1 or abcg2 indiï¬erent subpopulations of cells quantitative data in the histograms are presented as the mean fluorescent intensity from threeindependent experimentsculture p vs other groups at diï¬erent dxr concentrations figure2ato understand the relevant mechanism we measured theintracellular accumulation of dxr in cells by flow cytometrythe accumulation of dxr in cd44cd133 cells wasdetected as the lowest among these subpopulations at hrafter the exposure to μm dxr figure 2b we furtherfound that the intracellular accumulation of dxr in cd44cd133 cells was obviously increased by the treatment withverapamil an inhibitor for drug efflux cell membrane transporters of abcb1 and abcg2 figure 2b however theintracellular accumulation of dxr in cd44cd133 cellsdid not change by the treatment with bso a glutathione synthesis inhibitor that indirectly regulates drug efflux throughabcc1 figure 2b we also confirmed that the expressionof abcb1 p vs other groups but not abcg2 wasenhanced in cd44cd133 cells figure 2c suggestingthe probable role of abcb1 on dxr resistance in cd44cd133 cellsto further confirm the causal relationship between theenhanced drug efflux and dxr resistance we evaluatedthe cytotoxicity of dxr in the presence or absence of verapamil unexpectedly verapamil only partially enhanced thecytotoxicity of dxr in either cd44cd133 or cd44cd133 cells figure 3ait is well known that dxr interacts with nuclear dna toinhibit macromolecular biosynthesis therefore we alsoestimated the eï¬ect of verapamil on the nuclear accumulation of dxr the nuclear accumulation of dxr wasobserved obviously less in cd44cd133 than cd44cd133 cells but tended to have comparable levels withverapamil treatment figure 3b cd44cd133 cells showed better stress tolerance thancd44cd133 cells it is well known that the stress responsekinase p38mapk can be activated by various extracellularstresses and plays critical roles in cell survival and apoptosis although the basal level of phosphorylated p38mapkwas detected very similar between cd44cd133 andcd44cd133 cells p figure lower expressionwas observed in cd44cd133 than cd44cd133 cellsafter dxr exposure even under verapamiltreatmentp figure this suggests a better tolerance tostress of cd44cd133 cells independent on the accumulation of dxr 0cstem cells internationalˆ’ limaparev ytilibaiv llec limaparev ytilibaiv llec hoursŽŽŽŽ 𝜇mŽŽŽŽðœ‡mcd44cd133 cellscd44cd133ˆ’ cellsa hoursŽŽŽ ˆ’ limaparevstnuoccd44133ˆ’ cd44133𝜇mfl3hŽŽŽŽðœ‡m limaparevstnuocfl3hbfigure dxr resistance and nuclear dxr accumulation in cd44cd133 and cd44cd133 cells in the absence or presence of drug effluxinhibitor a mtt assay was done to compare the cytotoxicity of dxr in cd44cd133 and cd44cd133 cells with or without theaddition of μm verapamil data were expressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells b representative histograms of flow cytometry analysis showed the nuclear accumulation of dxr incells hr after the treatment by μm dxr with or without the addition of μm verapamil the results were reproducible in threeindependent experimentsverapamil ˆ’verapamil phosphop38 mapk totalp38 mapk noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ control hours dxr treatmentp p p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ hours dxr treatmentfigure diï¬erent expression of phosphorylated p38mapk between cd44cd133 and cd44cd133 cells representative blots andsemiquantitative data on the expression of phosphorylated p38mapk and total p38mapk in cells treated with μm dxr in theabsence or presence of μm verapamil the quantitative data are normalized to total p38mapk data are expressed as relative values tocd44cd133 cells without dxr treatment and presented as the mean ± sd from three independent experiments 0cstem cells internationaldxr ˆ’dxr ˆ’ limaparevstnuoc limaparevstnuoccd44133cd44133ˆ’stnuocfl1hfl1hstnuocfl1hafl1hverapamil ˆ’verapamil nrf2𝛽tubulin noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ controldxr treatment hours bp p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ dxr treatment hours figure diï¬erent antioxidant capacity between cd44cd133 and cd44cd133 cells a representative histograms of flow cytometryanalysis show the intracellular ros levels hr after the treatment by μm dxr in the absence or presence of μm verapamil theresults were reproducible in three independent experiments b representative blots and semiquantitative data on the expression of nrf2in cells treated with μm dxr in the absence or presence of μm verapamil the quantitative data are normalized to βtubulin dataare expressed as relative values to cd44cd133 cells without dxr treatment and presented as the mean ± sd from three independentexperiments cd44cd133 cells showed higher antioxidantcapacity than cd44cd133 cells it is also well known thatdxr generates ros and oxidative stress due to ros generation may induce the activation of p38mapk therefore weestimated the ros levels in cells with or without dxr exposure we observed a lower level of ros in cd44cd133 0cstem cells internationalstnuocstnuocstnuocstnuocstnuoc0nm ± stnuocfl2h5nm ± stnuocfl2h10nm ± stnuocfl2h15nm ± stnuocfl2h25nm ± stnuocfl2h0nm ± fl2h25nm ± ytilibaiv llecfl2h50nm ± fl2h75nm ± fl2h100nm ± fl2hŽŽŽŽðœ‡m cd44133ˆ’ cd44133 cd44133 control sirna cd44133 cd133 sirna abfigure continued 0cstnuo0cstnuo0cexpression of abcb10nm ± 0nmstnuo0c ± fl1h5nm ± fl1h50nmstnuo0c ± fl1hfl1h25nmstnuo0c ± stnuo0cfl1h100nm ± fl1hstem cells internationalaccumulation of dxrcontrol sirna 5nm ± fl3hcd133 sirna 5nm ± stnuo0cstnuo0cfl3hcdfigure the eï¬ect of silencing cd133 expression on dxr resistance of cd44cd133 cells a representative histograms of flowcytometry analysis on the expression of cd133 in cd44cd133 cells after silencing by diï¬erent dosages of targeted sirna quantitativedata in the histograms are presented as the mean fluorescent intensity from three independent experiments b mtt assay was done toevaluate the cytotoxicity to dxr cells were treated with nm sirna for hr followed by dxr treatment for another hr data areexpressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells crepresentative histograms of flow cytometry analysis on the expression of abcb1 in cells after silencing by diï¬erent dosages of targetedsirna quantitative data in the histograms are presented as the mean fluorescent intensity from three independent experiments drepresentative histograms of flow cytometry analysis on the accumulation of dxr quantitative data in the histograms are presented asthe mean fluorescent intensity from three independent experimentsthan cd44cd133 cells especially under dxr exposurebut verapamil did not obviously change the ros levelsfigure 5a based on these findings we speculated thatthe enhanced antioxidant capacity in cd44cd133 cellsmight help to maintain a lower level of phosphorylatedp38mapknrf2 a transcription factor that is well known to beactivated by oxidative stress such as ros and electrophilicsubstances can protect cells against various stresses we alsocompared the expression level of nrf2 between cd44cd133 and cd44cd133 cells western blotting showeda higher expression of nrf2 in cd44cd133 than cd44cd133 cells especially under dxr exposure p figure 5b and the enhanced expression of nrf2 in cd44cd133 cells was not cancelled by verapamil treatmentp figure 5b sirna treatment to further confirm the regulatory roleof cd133 in drug resistance we tried to silence cd133expression in cd44cd133 cells by sirna and then estimated cytotoxicity of dxr although the decrease ofcd133 expression was clearly observed by targeted sirnap vs nm figure 6a dxr resistance of cd44cd133 cells only partially improved figure 6b unexpectedlythe silencing of cd133 did not change theexpression of abcb1 in cd44cd133 cells even using 0cstem cells internationalexcessive concentrations of cd133 sirna p vs nmfigure 6c we also confirmed that the silencing of cd133did not aï¬ect the accumulation of dxr in cd44cd133cells p vs control sirna figure 6dthis suggests that beyond the drug excretion and redoxregulation other complex mechanisms are also likelyinvolved in the dxr resistance in cd44cd133 cells discussionby using the wellrecognized cell surface markers of cd44and cd133 for csc identification we tried to separate thehct8 human colon cancer cells into cd44 cd44 cd44cd133 and cd44cd133 subpopulations and then investigated how the expressions of cd44 and cd133 associatedwith drug resistance actually we checked several cancer celllines on the expression of cd44 and cd133 including helacells and a549 cells however both hela cells and a549 cellsshowed almost expression of cd44 only the hct8cells showed a partial expression of cd44 about anda rare expression of cd133 therefore we only isolateddiï¬erent subpopulations from hct8 cells for this studyfirst we found that the expression level of cd44 keptvery stable in the cd44cd133 cells but gradually declinedin cd44cd133 cells during a cell passaging process on theother hand some of cd44 cells shifted to express cd44 during a cell passaging process figure 1c these findings suggested the plasticity of cd44 expression in hct8 cellsactually ohata et al reported that cd44 highexpressedcells from human intractable colon cancer patients can diï¬erentiate into cd44 lowexpressed cells and a fraction of cd44lowexpressed cells can also generate cd44 highexpressedcells in a xenograft mouse model however it is unclearwhy the cd44cd133 cells but not cd44cd133 cellsstably maintain the expression level of cd44 unlike theextensive expression of cd44 with high plasticity the expression of cd133 was only observed in very few of the hct8cells with poor plasticitya number of previous studies have demonstrated thatcscs are likely resistant to chemotherapeutic drugs thecd44cd133 cells but not the cd44 and cd44cd133cells showed dxr resistance figure 2a according to thisdata the expression of cd133 but not cd44 seems to beclosely associated with drug resistance actuallythesecd44cd133 cells showed the enhanced expression ofabcb1 and the decreased intracellular accumulation ofdxr figures 2b and 2c liu et al reported that nonsmallcell lung cancer cells treated with lowdose cddp aresufficient to enrich cd133 cells and upregulate abcb1expression through notch signaling which thereforeincreases the crossresistance to dxr however theinhibition of abcb1 by verapamil only partially improvedthe dxr resistance of cd44cd133 cells in this studyto find other potential mechanisms involving in thedxr resistance of cd44cd133 cells we investigatedseveral interesting aspects including the stress protectionand redox regulation we found that p38mapk one of themost popular protein kinases known to be activated byinflammatory cytokines lipopolysaccharide osmotic shockultraviolet light and other stresses was more obviouslyinduced by dxr in cd44cd133 cells than cd44cd133cells figure moreover the activation of p38 mapk wasnot dependent on the intracellular accumulation of dxrfigure dxr is known to insert between the base pairs of dna oftumor cells and exhibits antitumor eï¬ects by suppressing thebiosynthesis of both dna and rna through the inhibitionof dna polymerase rna polymerase and topoisomeraseii reactions furthermore it is believed that dxr has theability to generate sufficient ros to raise oxidative stressindeed we observed dxrinduced ros generation in bothcd44cd133 and cd44cd133 cells butthe dxrinduced ros generation was detected even higher in cd44cd133 than cd44cd133 cells independent on the intracellular accumulation of dxr figures 5a 2b and 3bsuggesting the enhanced antioxidant capacity in cd44cd133 cellsthe keap1nrf2 control system plays a central role in theantioxidant defense mechanisms nrf2 is known as a transcription factor to activate various genes involving in biological defense mechanisms it has been reported that nrf2 isconstantly expressed in many cancer cells [“] moreover the enhanced expression of nrf2 has been confirmedto associate with poor prognosis of cancer patients [“]in our study nrf2 expression was detected higher in cd44cd133 than cd44cd133 cells and the diï¬erence in nrf2expression was observed even clearer between cells with dxradministrationindependent on the dxr accumulationfigure 5b these findings also clearly indicate theenhanced antioxidant capacity in cd44cd133 cellsalthough the absence of direct evidence by interferenceexperiment pathways involving in the stress protection andredox regulation might at least partially contributed to thedxr resistance of cd44cd133 cellsvery strangely our data showed that the silencing ofcd133 expression in cd44cd133 cells by sirna couldonly partially increase the cytotoxicity of dxr figure 6bbut did not change the expression of abcb1 and the intracellular accumulation of dxr figure 6c other unknownmechanisms beyond the drug excretion and redox regulationare asked to be defined on the dxr resistance of cd44cd133 cellsbased on data from the present study the expression ofcd133 rather than cd44 more closely associated with theresistance of cancer cells to anticancer drug as complexmechanisms including the drug excretion and redox regulation are likely involved in the drug resistance of cscs multiple approaches may be needed to overcome the big problemof drug resistance in cancer patientsabbreviationsabcb1abcg2atpbinding cassette subfamily b member 1pglycoproteinmultidrug resistance protein1mdr1atpbinding cassette subfamily g member2breast cancer resistanceproteinbcrpcd388 0cabcc1bsocscsdxrmttatpbinding cassette subfamily c member1multidrug resistanceassociated protein1mrp1buthionine sulfoximinecancer stem cellsdoxorubicinadriamycin345dimethylthiazol2yl25diphenyltetrazolium bromidenuclear factor erythroid 2related factor nrf2p38mapk p38 map kinaserossirnareactive oxygen speciessmall interfering rnadata availabilitythe data that support the findings of this study are availablefrom the corresponding author upon reasonable requestdisclosurethe funder played no role in the study design data collectionand analysis decision to publish or preparation of themanuscriptconflicts of interestthe authors indicate no potential conflicts of interestacknowledgmentsthis work was supported by a grantinaid for the ministryof education science sports culture and technology ofjapan grant numbers and 16k15622 and thecollaborative research program of the atomic bomb diseaseinstitute of nagasaki universityreferences r c elble œthe role of cancer stem cells in relapse of solidtumors frontiers in bioscience vol e4 no pp “ j e visvader œcells of origin in cancer nature vol no pp “ h clevers œthe cancer stem cell premises promises andchallenges nature medicine vol no pp “ y kinugasa t matsui and n takakura œcd44 expressed oncancerassociated fibroblasts is a functional molecule supporting the stemness and drug resistance of malignant cancer cellsin the tumor microenvironment stem cells vol no pp “ t ishimoto o nagano t yae et al œcd44 variant regulatesredox status in cancer cells by stabilizing the xct subunit ofsystem xc and thereby promotes tumor growth cancer cellvol no pp “ m tamada o nagano s tateyama et al œmodulation ofglucose metabolism by cd44 contributes to antioxidant statusand drug resistance in cancer cells cancer research vol no pp “ r c bates n s edwards g f burns and d e fisher œacd44 survival pathway triggers chemoresistance via lyn kinasestem cells internationaland phosphoinositide 3kinaseakt in colon carcinoma cellscancer research vol no pp “ l y w bourguignon k peyrollier w xia and e giladœhyaluronancd44 interaction activates stem cell markernanogandankyrinregulated multidrug efflux in breast and ovariantumor cells the journal of biological chemistry vol no pp “ stat3mediated mdr1expressiongene l y w bourguignon c earle g wong c c spevak andk krueger œstem cell marker nanog and stat3 signalingpromote microrna21 expression and chemoresistance inhyaluronancd44activated head and neck squamous cellcarcinoma cells oncogene vol no pp “ k tajima r ohashi y sekido et al œosteopontinmediatedenhanced hyaluronan binding induces multidrug resistance inmesothelioma cells oncogene vol no pp “ j ni p j cozzi j l hao et al œcd44 variant is associatedwith prostate cancer metastasis and chemoradioresistanceprostate vol no pp “ u carling l barkhatov h m reims et al œcan we ablateliver lesions close to large portal and hepatic veins with mrguided hifu an experimental study in a porcine modelblood vol no pp “ y wu and p y wu œcd133 as a marker for cancer stem cellsprogresses and concerns stem cells and development vol no pp “ u m gehling s erg¼n u schumacher et al œin vitro diï¬erentiation of endothelial cells from ac133positive progenitorcells blood vol no pp “ m peichev a j naiyer d pereira et al œexpression ofvegfr2 and ac133 by circulating human cd34 cellsidentifies a population of functional endothelial precursorsblood vol no pp “ n uchida d w buck d he et al œdirect isolation of humancentral nervous system stem cells proceedings of the nationalacademy of sciences of the united states of america vol no pp “ b j cummings n uchida s j tamaki et al œhuman neuralstem cells diï¬erentiate and promote locomotor recovery inspinal cordinjured mice proceedings of the national academy of sciences of the united states of america vol no pp “ b bussolati s bruno c grange et al œisolation of renal progenitor cells from adult human kidney the american journalof pathology vol no pp “ l riccivitiani d g lombardi e pilozzi et al œidentification and expansion of human coloncancerinitiating cellsnature vol no pp “ c a o™brien a pollett s gallinger and j e dick œa humancolon cancer cell capable of initiating tumour growth inimmunodeficient mice nature vol no pp “ l lin a liu z peng et al œstat3 is necessary for proliferation and survival in colon cancerinitiating cells cancerresearch vol no pp “ n haraguchi m ohkuma h sakashita et al œcd133cd44 population efficiently enriches colon cancer initiating cellsannals of surgical oncology vol no pp “ 0cstem cells international d inoue t suzuki y mitsuishi et al œaccumulation ofp62sqstm1 is associated with poor prognosis in patientswith lung adenocarcinoma cancer science vol no pp “ j q ma h tuersun s j jiao j h zheng j b xiao anda hasim œfunctional role of nrf2 in cervical carcinogenesis plos one vol no article e0133876 q yang h deng h xia et al œhigh nfe2related factor expression predicts poor prognosis in patients with lung cancer a metaanalysis of cohort studies free radical researchvol pp “ s sarvi a c mackinnon n avlonitis et al œcd133 cancerstemlike cells in small cell lung cancer are highly tumorigenicand chemoresistant but sensitive to a novel neuropeptideantagonist cancer research vol no pp “ q zhang s shi y yen j brown j q ta and a d le œasubpopulation of cd133 cancer stemlike cells characterized in human oral squamous cell carcinoma confer resistanceto chemotherapy cancer letters vol no pp “ s ma t k lee b j zheng k w chan and x y guanœcd133 hcc cancer stem cells confer chemoresistance bypreferential expression of the aktpkb survival pathwayoncogene vol no pp “ s bao q wu r e mclendon et al œglioma stem cells promote radioresistance by preferential activation of the dnadamage response nature vol no pp “ c yan l luo c y guo et al œdoxorubicininduced mitophagy contributes to drug resistance in cancer stem cells fromhct8 human colorectal cancer cells cancer letters vol pp “ s goto y ihara y urata et al œdoxorubicininduced dnaintercalation and scavenging by nuclear glutathionestransferase ϝ the faseb journal vol no pp “ h ohata t ishiguro y aihara et al œinduction of the stemlike cell regulator cd44 by rho kinase inhibition cont
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" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to 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 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the œconsensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from ˆ’ to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as œphagosomeœautoimmune thyroid disease œantigen processing andpresentation œb cell receptor signaling pathway œintestinal immune network for iga production œinflammatorybowel disease œprimary immunodeficiency œth1 andth2 cell differentiation œth17 cell differentiation œnatural killer cell mediated cytotoxicity and œnfˆ’kappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ “] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ “] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [“] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors™ contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didn™t involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am “biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw “simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol “huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem “liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg “xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett “zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med “ binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med“ yoshihara k shahmoradgoli m martínez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol “ deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med “ pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a
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"There are two kinds of localizing procedures: marking with thoracoscopically directly visible materials and marking with radio-opaque materials. Examples of directly visible materials are hook wire methylene blue and indocyanine green. Ethiodized oil (lipiodol) barium and iodine contrast agents are used for radio-opaque markers. Each marking method has strong and weak points. Localization with a hook wire is easy to perform but carries a high risk of pneumothorax and a propensity to dislodge during transport and surgical preparation (6 7). Methylene blue and indigo carmine have a tendency to diffuse over a large area by the time the operation is done and render localization features inadequate (8 9). The use of a radio-opaque marker (such as barium or lipiodol) requires an intraoperative fluoroscopy to confirm an adequate excision as well as lead to increased radiation exposure (10-13). The use of mixture has been reported to make up for the weakness of marking materials. For example the problem of dye diffusion has led to attempts to use a mixture of dye with various materials such as cyanoacrylate adhesive or collagen or autologous blood (14-16). However they have not been widely used for localization due to difficulties in making and manipulation. Lipiodol and methylene blue are commonly used materials for localization (17-20). We hypothesized that lipiodol reduces the spread of methylene blue and provides additional localization opportunities by its radio-opacity. The use of a mixture of lipidol and methylene blue (MLM) for a percutaneous injection material requires a high success rate for appropriate localization and a low complication rate. To our knowledge there have been no reports that evaluate the availability of MLM as a percutaneous injection material in human lungs. This study compared MLM with methylene blue as a percutaneous injection material for pulmonary localization in rabbit lungs. MATERIALS AND METHODS Animal preparation This study was performed after approval by the Institutional Animal Care and Use Committee (IACUC) in Seoul National University Hospital biomedical research institute (IACUC approval No. 11-0356). Twenty-four adult New Zealand White rabbits were used. We recorded their weight before the procedures. The animals were randomly divided into two groups: Group A (n=12) and Group B (n=12) each sacrificed at about 6 hr and 24 hr after percutaneous injections respectively (Fig. 1). Six hours after percutaneous injections were same day operations of the preoperative localization; and 24 hr after percutaneous injections were next day operations of the preoperative localization. The injection of each material was done in all 24 subjects because we injected methylene blue and MLM at two different lung sites for each subject. Percutaneous injection materials: mixture of lipiodol and methylene blue versus methylene blue A pilot study was performed to decide the optimal amount of materials for percutaneous injections. Methylene blue (1% 100 mg/mL TERA Pharmaceuticals Buena Park CA USA) of 0.3 to 0.9 mL was used for human lung localization in previous studies by Wicky et al. (18) and Vandoni et al. (19). In the pilot study with rabbit lungs we injected 0.1 mL and 0.05 mL of methylene blue and MLM in four subjects. We found that staining was extensive (more than half height of one lobe) with 0.1 mL and localized (about 1 cm of staining diameter) with 0.05 mL for both methylene blue and MLM. Extensive dispersion made it difficult to find exact injecting sites; subsequently 0.05 mL of methylene blue was administered. We made variable mix ratios of lipiodol and methylene blue in vitro; 1:1 1:2 1:3 1:4 and 1:5 in order to find an appropriate mixing ratio of lipiodol (480 mg Iodine/mL Andre Guerbet Aulnay-sous-Bois France) and methylene blue. The separation of two materials occurred instantly after mechanical blending to the fat-soluble character of lipiodol and the water-soluble character of methylene blue. A higher concentration of lipiodol in MLM resulted in increased uneven blending and rapid separation. A mixture with a 1:6 (or lower) mixing ratio contained a minimal amount of lipiodol and it might make it difficult to be detected on the fluoroscopy; subsequently we decided that 1:5 was an appropriate mixing ratio for injection. A total of 0.06 mL of MLM (0.01 mL of lipiodol plus 0.05 mL of methylene blue) was administrated in each subject to avoid the effect of different volumes of methylene blue to the diffusion extent of the materials. CT guided percutaneous injections Percutaneous injection was performed with computed tomography (CT) guidance (Discovery CT750 HD; GE Healthcare Waukesha WI USA). We performed pre-procedural CT scans in order to determine an appropriate skin entry site for the successful placement of a needle in the desired location. The desired location was the basal portion of both caudal lobes around the mid-scapula line. We tried to situate the needle tip at 5 mm depth from the visceral pleura and avoid passing through the pulmonary vessels. We placed the needle of 20 gauze and 3.5 cm length in the lung parenchyma after marking the appropriate skin entry site. The parameters of CT used in our study were: tube voltage of 120 kV tube current of 25 mA slice thickness of 2 mm thickness and gantry rotation speed of 350 milliseconds. We connected 1 mL syringe to the needle hub and retracted the syringe piston to confirm that no blood was aspirated after the needle tip was accurately located within the desired location. We then injected the materials and immediately removed the needle. On the procedural CT scan we measured the distance from the skin-entry to the needle tip and the depth from visceral pleura to the needle tip. A post-procedural CT scan identified procedure-related complications that included the leakage of injecting materials and pneumothorax; in addition we recorded the extent shape and density of radio-opacity of MLM after injection. The extent of MLM was defined as a maximum diameter of the radio-opacities. The shape of radio-opacity was categorized into 3 groups (small faint nodular scattered nodular and discrete compact nodular). We recorded the injection time to measure the time interval between injection and sacrifice. Fluoroscopic examinations A successful localization of lipiodol was determined by fluoroscopic examination; subsequently we evaluated the radio-opacity of MLM using the fluoroscopy X-ray unit (BV Pulsera; Philips Medical Systems Best The Netherlands) at the immediate post-procedure session and the follow up session at 6 hr in Group A and 24 hr in Group B. The parameters of fluoroscopy were: tube voltage of 59 kV and tube current of 946 mA. We obtained anteroposterior fluoroscopic images of the thorax of the rabbit with a 17 cm of field of view. A radio-opaque ruler of 5 cm was located near the rabbit in order to estimate the exact size of lipiodol opacity. We recorded the time of the fluoroscopic examinations and the radiographic findings of MLM (size and shape of the radio-opacity). Evaluation of the staining and radio-opacity We assessed the directly visible staining on the freshly excised lung surface and radio-opacity of MLM on the fluoroscopic examinations using 4-point scoring in order to compare the localization ability of MLM and methylene blue as a percutaneous injection material. A blind reviewer who was unaware of the injection materials assessed the staining ability. In order to evaluate the staining ability the blind reader reviewed the photographic images of the freshly excised lung specimens obtained before formalin fixations and rated the staining by 4-point scores: 0=non-visualization of staining 1=inappropriate; extensive dispersion made it difficult to find accurate injecting locations 2=acceptable; available to estimate injecting locations in spite of the dispersion and 3=excellent definitely localized staining (Fig. 2). The maximum diameter of the staining extent on the lung surface was measured. We calculated and compared scores and extent of staining between two materials. For the fluoroscopic findings the radio-opacity of MLM was evaluated using 4-point scoring: 0=no detectable radio-opacity 1=inappropriate minimally increased opacity 2=acceptable low density of increased opacity 3=excellent compact nodular increased opacity (Fig. 3). We compared the average scores of initial and follow up fluoroscopic examinations. We considered a score of 0 or 1 as inappropriate and a score of 2 or 3 as appropriate for localization for both staining and radio-opacity. We compared the number of appropriate or excellent localization between MLM and methylene blue. Sacrifice and histopathologic examinations Both freshly excised entire lungs were used as final specimens. The lung tissues were fixed in 10% neutral formalin embedded in paraffin and cut into 5 µm thick slices after we took photographs to record staining on the lung surface. We made 4 axial slices that covered the center of the staining. The slices were subjected to hematoxylin-eosin (H-E) stain to the evaluate lung parenchymal change. We evaluated the presence or absence of neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus. The extent of each histopathologic finding was estimated using visual grading scores as 0 (no) 1 (focal) or 2 (diffuse). Localized parenchymal change (<50% of total area) surrounded by normal lung was defined as focal. Extensive lung parenchymal change (?50% of total area) that replaced normal lung was defined as diffuse. An experienced pathologist with eight years of experience reviewed all slices. The overall severity of the lung parenchymal change was defined as a total score by adding visual grading scores for each histopathologic finding. We compared the overall severity score between MLM and methylene blue as well as between Group A and Group B. Statistical analysis All data are expressed as mean±standard deviation (SD) unless otherwise stated. Comparisons of the average scores were performed by two-tailed unpaired Student's t-test or Mann-Whitney test. We used a Fisher's exact test to compare the number of subjects in the subgroups. Linear by linear association evaluated the association of the extent of lung parenchymal change and materials or groups. Null hypotheses of no difference were rejected if the P values were less than 0.05. The statistical analysis was performed with commercially available statistical software IBM SPSS Statistics version 20.0 (IBM Corp. in Armonk NY USA). RESULTS Subject characteristics procedural records time interval of injection and examinations Among the 24 subjects included in our study successful CT-guided percutaneous injections into the desired location of the lung were achieved in 21 subjects (11 in Group A and 10 in Group B). Three subjects died during anesthesia. Mean weight was 3.2±0.2 kg for Group A and 3.3±0.2 kg for Group B. Injection depth from visceral pleura to needle tip was 0.4±0.1 cm (range: 0.3-0.6 cm) for MLM and 0.4±0.1 cm (range: 0.3-0.7 cm) for methylene blue (P=0.43). Distance from skin to needle tip was 2.8±0.6 cm (range: 2.1-5.0 cm) for MLM and 2.8±0.3 cm (range: 2.2-3.5 cm) for methylene blue (P=0.83). Of 42 CT-guided percutaneous injections total number of procedure related complications was 10 (24%) including 7 leakage (all in MLM) and 3 pneumothorax (2 in MLM 1 in methylene blue). The complication rate in MLM was significantly higher than methylene blue (43% vs 5%) (P=0.004). On post-procedural CT images the extent of the radio-opacity of MLM was"
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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 Pearson™s correlation test and principal component analysis Differentially expressed genes DEGs between normal and PCRC were identified and the protein“protein 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 person™s 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 GSE81558Verification for repeatability of intragroup dataFirst repeatability of intragroup data were verified by the Pearson™s 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 ‰¤ ˆ’ 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 GSE81558Protein“protein 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 KEGGSignificant 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 Chin™s [] 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 genesPearson™s 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 fluid 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 ˆ’ 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 Kaplan“Meier 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 Pearson™s 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 Pearson™s 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 peptidyl“threonine 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 2DSignificant module network and identification 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 Pearson™s 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 Kaplan“Meier 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 S5B“D 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 identification 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 Kaplan“Meier 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 symbolPearson™s correlation coefficientPvalueρaCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2Bˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’PvalueaPearson™s correlation coefficient between PCRC and relevant characteristics ρ Pearson™s correlation coefficientbMultiple linear regression analysis PCRC primary colorectal cancer P005 P001 P0001Table Receiver operator characteristic curve analysis of hub gene expression for PCRCGene symbolPCRCCLCA4GUCA2AGCGSSTMS4A12PLP1CHGAPYYVIPGUCA2BAUC0987maxPvalue95CI““““““““““AUC area under curve max the maximum of AUC Significant variables ODT Optimal diagnostic thresholdPCRC primary colorectal cancer P0001Correlation linear regression and ROC analysisThe Pearson™s correlation coefficient was used in the correlation analysis and CLCA4 ρ ˆ’ P0001GUCA2A ρ ˆ’ P0001 GCG ρ ˆ’ P0001 SST ρ ˆ’ P0001 MS4A12 ρ ˆ’P0001 PLP1 ρ ˆ’ P0001 CHGA ρ ˆ’ P0001 PYY ρ ˆ’ P0001 VIP ρ ˆ’ P0001 and GUCA2B ρ ˆ’ 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‰¥ monthsPearson™s chisquared test was usedP005Figure The verification 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 Kaplan“Meier 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 body™s 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 epithelial“mesenchymal 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
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" inflammatory pseudotumour has been used to describe an inflammatory or fibrosing tumoral processof an undetermined cause that may involve a variety of an systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potential for recurrence and persistent local growth in this we report a patient with a big mass of uncertain nature and behaviorcase presentation a 60yearold woman presented with a 1week history of abdominal pain fever and jaundicesix months before she had had right upper quadrant pain that was interpreted as biliary colic a contrastenhancedct scan showed a big mass of soft tissue with diffuse infiltration of the gallbladder displacement of the transversecolon hepatic flexure and duodenum for diagnostic distinction between a chronic inflammatory disease or aneoplasm exploratory laparotomy was required intraoperative exploration disclosed a big mass of hard textureinvolving the gallbladder with multiple concrements hepatoduodenal ligament right and transverse mesocolonstomach and duodenumcholecystectomy was performed preserving adjacent ans with macroscopic desmoplastic reactionhistopathologic examination of the gallbladder showed a spindle cell proliferation with diffuse chronicinflammatory infiltrate of lymphocytes plasma cells and hyalinized fibrous stroma no vascular invasion or cellularatypia were evident inflammatory pseudotumour is a rare condition and diagnostic distinction from a chronicinflammatory disease or other neoplasm is only possible by histopathologic examination there is a limited numberof case reports in the literature indicating tumor location in the gallbladderkeywords inflammatory pseudotumor gallbladder inflammatory pseudotumour is a rare lesion that hasbeen described in various ans and tissues intraabdominal variants of the disease are reported to occurmost frequently in the liver spleen mesentery and extrahepatic bile duct the location of the gallbladder iseven more uncommon correspondence acd3202yahooesdepartment of surgery and pathology puerto real university hospital c¡dizspainmalignant transformations and recurrences of inflammatory pseudotumour have been reported years aftersurgery therefore longterm followup is necessary evenfor patients successfully treated by surgical resectionelevated igg4 serum levels have been reported in association with this illness as well as abundant igg4 positivity in tumor infiltrating plasma cells signs suggestiveof an igg4related disease a high serum igg4 concentrations thus provides a useful means of distinguishingthis disorder from other differential diagnoses 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 0ccalvo bmc gastroenterology page of pharmacologic treatments have also been reported forigg4associated inflammatory pseudotumor and thereare even cases of complete resolution of the disease withsteroids treatments however in the presented case thiscondition did not occur so treatment was exclusivelysurgicalcase presentationa 60yearold woman presented to the emergencyroom with abdominal pain fever pruritus and jaundicesince week the patient had a history of smoking anda family history of pancreatic canceron physical examination a hard and painful mass wasidentified on the right hypochondrium blood laboratoryexamination showed extrahepatic cholestasis enzymestotal bilirubin mgdl direct bilirubin mgdlast ul alt ul ggt ul ldh ul alkaline phosphatase ul in anamnesis thepatient referred to abdominal pain occurring during thelast months located in the right upper quadrant whichhad been interpreted as biliary colic by her generalpractitionertumour markers and blood count showed no alterations viral serology autoimmunity antibodies metanephrines and urine normetanephrine were within thenormal rangea large mass associated with the gallbladder was identified by abdominal ultrasound contrastenhanced ctscan disclosed a large soft tissue mass originating fromthe gallblader with homogenous contrast enhancementand without clear infiltration of the hepatic parenchymathe mass displaced the transverse colon hepatic flexureand duodenum no lymphadenopathies were identifiedin the hepatoduodenal ligament pancreas retroduodenum or celiac axis fig 1a b the gallbladder was distended contained stones and had a regular lumenwhile there was slight dilatation of the intrahepatic bileduct on magnetic resonance imaging fig 1csmallerwith these findings a diagnostic distinction between a chronic inflammatory disease or a neoplasticprocess was necessary the biopsy of the mass wasperformed under ultrasonographic control histopathologic examination showed spindle cells and some inflammatory cells ofsize and absence ofxanthic cells the tumor showed a mesenchymal aspect that was confirmed by absence of epithelial cellson pancytokeratin staining while some histiocyteswere recognized in summary the pathology diagnosiswas a mesenchymal process that could be reactive ormalignant the microbiological study of the bile obtained from gallbladder punctured showed a nonpurulent gram™s stain and negative cultures for bothaerobic and anaerobic germs the oral endoscopy andbiopsies of the second part of the duodenum didn™tshow any pathological conditionexploratory laparotomy was decided and cholecystectomy could be performed preserving the adjacent ans with macroscopic desmoplastic reaction the masswas peeled off the transverse colon first and the secondpart of the duodenum and common bile duct fig the histopathological examination offibrousspecimen disclosed sclerosingthe resectiontissue withfig axial and coronal computed tomography images showing a large mass of diffuse soft tissues originating from the gallbladder anddisplacing the duodenum transverse colon and hepatic flexure a b in magnetic resonance imaging the gallbladder was distended andcontained stones associated with a slight dilatation of the intrahepatic bile duct c 0ccalvo bmc gastroenterology page of stains were performedto achieve a definitive classification complementaryimmunohistochemicalandshowed positive staining for smooth muscle actin in themuscular layer ofthe gallbladder and vessel wallscd34 in the vascular lumen cd68 in histiocytes andremained negative for anaplastic lymphoma kinasealk and pancytokeratin panck masson™s trichrome stain showed intense positivity on collagen fibers less than of the tumor cells sample were ki67positive and plasma cells were igg4 positive per highpower field taken together these findings confirmed thediagnosis of inflammatory pseudotumor of the gallbladder with sclerosing cholangitis associated with a normallevel of serum of immunoglobulin g4 of mgdl “ mgdlno local recurrence was detected at the threeyearsfollowup on ct scandiscussion and sthe term inflammatory pseudotumour has been used todescribe an inflammatory or fibrosing tumoural processof undetermined cause that may involve a variety ofan systems including the lungs spleen liver lymphnodes pancreas and extrahepatic bile duct with potentialfor recurrence and persistent local growth [ ] thereis a limited number of case reports in the literature indicating gallbladder location [ ]fig on laparotomy a large and hard mass was identified in thegallbladder with stones displacing but not infiltrating right andtransverse mesocolon stomach duodenum andhepatoduodenal ligamenthistiocytes chronic lymphocytic inflammatory infiltrateand plasma cells with isolated eosinophils and no epithelial malignancy the mass presented as an expansivegrowth from the outer portion of the muscular layer ofthe gallbladder to the surrounding fatty tissuethe definitive pathological diagnosis was inflammatorypseudotumour of the gallbladder with chronic sclerosingcholangitis fig a bfig histopathologic examination disclosed a thickened gallbladder wall a with spindle cells and proliferation of connective fibrous tissuewithout signs of celular atypia b and inflammatory cells including lymphocites plasma cells and hyalinized fibrous tissue without vascularinvasion c few plasma cells were igg4 positive in relation to the whole inflammatory cell infiltrate d 0ccalvo bmc gastroenterology page of consent for publicationwe confirm in this statement that a written consent to publish thisinformation was obtained from study participant and the proof of consentto publish from study participants can be provided at any timethe authors have in their possession the informed consent of the patientbiomed central consent formcompeting intereststhe authors declare that they have no competing interestsreceived january accepted august referencesbehranwala ka straker p wan a fisher c thompson jn inflammatorymyofibroblastic tumour of the gallbladder world j surg oncol sinha l hasan a sngh ak bhadani pp jha an singh pk kumar minflammatory myofibroblastic tumor involving liver gallbladder pylorus andduodenum a rare case presentation int j surg case rep “badea r veres aa andreica v inflammatory myofibroblastic tumor ofthe gallbladder imaging aspects j med ultrason “abrantes cf silva mr oliveira rc eloy c cipriano ma castro lpinflammatory myofibroblastic tumour arising incidentally as a polypoidlesion in the gallbladder j bras patol med lab v51 n p “koea jb broadhurst gw rodgers ms inflammatory pseudotumor ofthe liver demographics diagnosis and the case for nonoperativemanegement j am coll surg “sato y kojima m takata k immunoglobulin g4relatedlymphadenopathy with inflammatory pseudotumorlike features med molmorphol “hamano h kawa s horiuchi a high serum igg4 concentrations inpatients with sclerosing pancreatitis n engl j med “aldhahab h mcnabbbaltar j albusafi s barkun an immunoglobulin g4related pancreatic and biliary disease can j gastroenterol “lee ys lee sh lee mg immunoglobulin g4related diseasemimicking unresectable gallbladder c¡ncer gut liver “ muduly d deo sv shukla nk inflammatory myofibroblastic tumor ofgall bladder trop gastroenterol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsinflammatory pseudotumour appear to be more common in noneuropean populations they usually occur ininfancy and young adults but can occur in the elderly []the igg4 serum level should be determined due to acommon association of elevated serum igg4 with this illness and some authors describe abundant igg4 positivityin plasma cells as suggestive of igg4related disease []high serum igg4 concentrations might provide afromuseful means of distinguishing this disorderother lesions [“]the histopathological examinations showed sclerosingfibrous tissue with histiocytes chronic lymphocytic inflammatory infiltrate and plasma cells with isolated eosinophils compatible with igg4related disease howeveras igg4 serum levels were within normal range and theigg4 tissue expression was very weak there is no strongevidence of a clear association with igg4related diseaseinflammatory pseudotumour can generally be considered to be a relatively rare disease of undefined originwith a great variety of symptoms causing diagnosticchallenges in the distinction of chronic inflammatorydisease and neoplasminflammatory pseudotumoris defined as nonneoplastic but is currently considered as a tumour withlowgrade malignant transformations it has been reported in the liver urinary bladder kidney breast stomach pancreas spleen and retroperitoneum there is alsoa limited number of case reports in the literature indicating the gallbladder location these patients must beobserved with close and regular longterm followup asrecurrences have been reported to occur four to yearsafter surgery []acknowledgementsangela hens head of the pathology department of puerto real universityhospital for providing her pathological knowledgemario bruno for providing the new corrections in the translation of thismanuscriptpd dr med ulrich f wellner consultant surgeon pancreatic surgery andresearch clinic of surgery uksh campus l¼beck germany for providingthe latest english language correctionsauthors™ contributionsac the first author directed the operation and wrote the paper js hasparticiped in the design of the report and copy edited the manuscript adhas participated in the operation mc has participated in the operation gmhas made the pathological diagnosis and part of the literature review allauthors read and approved the final version of the manuscript all authors ofthis manuscript are in agreement with its content and are not beingpublished or under consideration in another scientific journalfundingnot applicableavailability of data and materialsdata sharing is not applicable to this as no data sets were generatedor analysed during the current studyethics approval and consent to participatenot applicable 0c"
0
"Identification of key differentially expressed mRNAs and microRNAs in non‘small cell lung cancer using bioinformatics analysisWEIWEI WANG SHANSHAN WANG and LEI PANDepartment of Pulmonary and Critical Care Medicine Beijing Shijitan Hospital Capital Medical University Beijing PR ChinaReceived September Accepted January 103892etm20209105Abstract Nonsmall cell lung cancer NSCLC is a leading cause of mortality worldwide However the pathogenesis of NSCLC remains to be fully elucidated Therefore the present study aimed to explore the differential expression of mRNAs and microRNAs miRNAsmiRs in NSCLC and to determine how these RNA molecules interact with one another to affect disease progression Differentially expressed genes DEGs and differentially expressed miRNAs DEMs were identified from the GSE18842 GSE32863 and GSE29250 datasets downloaded from the Gene Expression Omnibus GEO database Functional and pathway enrichment analysis were performed based on Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG databases STRING Cytoscape and MCODE were applied to construct a proteinprotein interaction PPI network and to screen hub genes The interactions between miRNAs and mRNAs were predicted using miRWalk and a miRNAmRNA regulatory network was constructed The prognostic value of the identified hub genes was then evaluated via KaplanMeier survival analyses using datasets from The Cancer Genome Atlas A total of DEGs and DEMs were identified from the GEO datasets The enriched pathways and functions of the DEGs and target genes of the DEMs included osteoclast differentiation cell adhesion response to a drug plasma membrane extracellular exosome and protein binding A subnetwork composed of genes was extracted from the PPI network and the genes in this subnetwork were mainly involved in the cell cycle cell division and DNA replication A miRNAgene regulatory network was constructed with miRNAgene pairs based on DEMs and DEGs KaplanMeier survival analysis Correspondence to Professor Lei Pan Department of Pulmonary and Critical Care Medicine Beijing Shijitan Hospital Capital Medical University Tieyi Road Haidian Beijing PR ChinaEmail panleibjsjthcnKey words nonsmall cell lung cancer bioinformatics microRNA miRNAmRNA network KaplanMeier plotindicated that the expression of ubiquitin E2 ligase C cell division cycle protein DNA topoisomerase IIα aurora kinase A and B cyclin B2 maternal embryonic leucine zipper kinase slit guidance ligand phosphoglucomutase endomucin cysteine dioxygenase type dihydropyrimidinaselike miR‘130b miR‘ and miR‘ was significantly associated with overall survival of patients with lung adenocarcinoma In the present study a miRNAmRNA regulatory network in NSCLC was established which may provide future avenues for scientific exploration and therapeutic targeting of NSCLCIntroductionLung cancer is one of the most common types of cancer and is the leading cause of cancerassociated mortality worldwide accounting for of all deaths due to cancer Approximately of lung cancers are nonsmall cell lung cancer NSCLC In turn NSCLCs are comprised of lung adenocarcinoma LUAD of lung cancers lung squamous cell carcinoma LUSC of lung cancers and large cell carcinoma of lung cancers Despite advances in diagnostics surgery and medication in recent decades the average 5year survival rate of patients with NSCLC remains as low as This poor prognosis is a consequence of high rates of tumor metastasis and recurrence and numerous signaling pathways having been identified to be involved in these processes Thus an enhanced understanding of the molecular mechanisms controlling NSCLC progression is required to improve the low survival rate The development of highthroughput sequencing has allowed for comprehensive comparisons of gene expression profiles thereby identifying differentially expressed genes DEGs between tumor and normal tissues Changes in expression levels usually indicate pathological states as proteins encoded by DEGs may be involved in tumorigenesis and tumor progression microRNAs miRNAsmiRs are short noncoding RNA molecules that mediate the posttranscriptional regulation of mRNAs via binding to complementary sequences in the 'untranslated region UTR of mRNAs and suppressing their translation or mediating their degradation An individual miRNA may regulate hundreds of different mRNA molecules highlighting the existence of miRNAmRNA regulatory 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCnetworks Depending on how it is expressed a specific miRNA may therefore act to suppress or promote oncogenesis via specific effects on relevant target mRNAs Indeed miRNA profiling efforts have been used to identify specific miRNA signatures associated with particular tumor subtypes thereby allowing for cancer diagnosis treatment planning and prediction of patient prognosis Bioinformatics analysis of gene expression microarray data provides a useful tool for revealing numerous previously unrecognized mRNAs and miRNAs that may be implicated in the pathogenesis of cancer or other diseases In the present study gene expression datasets were analyzed using an integrated bioinformatics approach in order to identify DEGs and differentially expressed miRNAs DEMs between NSCLC tumors and healthy control tissues Functional enrichment and proteinprotein interaction PPI network analyses were performed to better establish the functions of these mRNAs and miRNA and these approaches were combined with an analysis of mRNAmiRNA interactions to screen hub genes and miRNAs in this regulatory network Through this approach the present study aimed to further elucidate the molecular mechanisms of NSCLC to identify potentially novel therapeutic strategies for its treatmentMaterials and methods Microarray data information The datasets used in the present study were obtained from the Gene Expression Omnibus GEO database www ncbinlmnihgovgeo The original gene expression profiles from the datasets GSE18842 GSE32863 and GSE29250 were used and the clinical information of the patients was obtained from the original s The GSE18842 dataset included samples tumors and controls and all samples were paired except tumors and control The platform used for the GSE18842 dataset was GPL570 HGU133_Plus_2 Affymetrix Human Genome U133 Plus Array The GSE32863 dataset included samples tumors and controls and all samples were paired The platform used for the GSE32863 dataset was the GPL6884 Illumina Human WG6 v30 expression beadchip The GSE29250 dataset included samples tumors and controls and all samples were paired The platform used for the GSE29250 dataset was the GPL8179 Illumina Human v2 MicroRNA expression beadchipIdentification of DEGs To compare gene expression profiles the GEO2R tool httpwwwncbinlmnihgovgeogeo2r accessed March which is based on the limma package in R was used to individually identify DEMs and DEGs in each dataset To control for type I error as a result of multiple comparisons within each dataset the false discovery rate FDR determination feature automatically included in the GEO2R tool was employed Significant DEGs were those that remained significant after FDR correction when tested via multiplecomparisons ttests and fold change FC2 and P005 were set as the cutoff criteria Any probes that did not correspond to a specific gene symbol were then filtered from the resultant dataGene function analysis For gene ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathway analyses of DEMs and DEGs the database for annotation visualization and integrated discovery DAVID database v68 httpdavidabccncifcrfgov was used focusing specifically on humans and using all genes as an enrichment background Significant enrichment in these analyses was determined based on an adjusted Pvalue of as established via the BenjaminiHochberg method These Pvalues were determined on the basis of a cumulative hypergeometric distribution calculating qvalues based on the BenjaminiHochberg procedure as a means of controlling for multiple testing For comparisons of hierarchical clustering of enriched terms clusters were designated as groupings that had a similarity score with the most significant term within a given cluster being selected to represent the cluster as a wholePPI network construction and analysis The online Search Tool for the Retrieval of Interacting Genes and proteins STRING database v110 stringdborg was used for PPI network construction PPI pairs with a combined score ‰¥ were used to generate the network Cytoscape v340 cytoscapeorg was used to visualize the regulatory interactions between these genes and CentiScaPe v22 Center for Biomedical Computing University of Verona Italy was used to analyze network distributions based on topological properties The Molecular Complex Detection application MCODE v16 was used to identify and extract subnetworks from the global PPI network based on the kcore algorithm The genes with a degree ‰¥ in this regulatory network were identified as hub genes as described previously Prediction of the mRNA‘miRNA interactions An online tool called miRWalk httpmirwalkummuniheidelbergde which integrates predictive outputs of TargetScan and miRDB was used to predict DEG and DEM interactions A score ‰¥ was considered as the critical criterion for the miRWalk predictive analysis Only the target mRNAs predicted by all tools miRWalk TargetScan and miRDB were used for further analysis By overlaying identified DEGs and these predicted mRNA targets a miRNAmRNA regulatory network was constructed and then visualized using CytoscapeAnalysis of datasets from the cancer genome atlas TCGA TCGA is an online database that may be used to research and explore publicly available datasets cancergenomenihgov accessed March including RNA sequencing RNAseq data from TCGA samples of different types of cancer In the present study the tumor types were limited to LUAD and LUSC RNAseq data and clinical data from LUAD and LUSC samples from TCGA datasets were used Based on the approach previously outlined by Li and Dewey a PERL program was used to multiply the ˜scaled estimate™ by yielding transcripts per million TPM values for all gene expression as TPM values were thought to be a more reliable means of comparing gene expression than fragments per kilobase of TPMmapped reads or reads per kilobase of TPMmapped reads values In the present study to improve the reliability of the analysis the expression of hub genes was validated in TCGA datasets using Gene Expression Profiling Interactive Analysis GEPIA v10 httpgepiacancerpkucn For each of the hub genes patients were 0cEXPERIMENTAL AND THERAPEUTIC MEDICINE Figure Volcano plots and clustering heat maps of the differentially expressed genes between the non small cell lung cancer and normal tissues A1 Volcano plots of differentially expressed mRNA from the GSE18842 dataset A2 Volcano plots of differentially expressed mRNA from the GSE32863 dataset A3 Volcano plots of differentially expressed miRNA from the GSE32863 dataset Each graph represents the expression of the gene plotted by log2 fold change on the horizontal axis and log10q value on the vertical axis Red represents upregulation green represents downregulation and blue represents no significant difference B1 Clustering heat maps based on GSE18842 dataset B2 Clustering heat maps based on the expression of mRNAs in GSE32863 dataset B3 Clustering heat maps based on the expression of miRNAs in GSE32863 dataset Orange represents upregulation and blue represents downregulation miR microRNA hsa Homo sapiens UP upregulation DW downregulation NoDiff no difference NA not available Some miRNAs in the databases have probes but no datastratified into groups based on expression levels of each gene and differences in patient survival were analyzed generating hazard ratio HR and CI values as well as logrank Pvalues for each comparison ResultsIdentification of DEGs and DEMs In the GSE18842 and GSE32863 datasets and DEGs were identified respectively of which and were upregulated and and were downregulated Fig 1A and B A total of DEGs were shared between these datasets upregulated and downregulated The GSE32863 dataset yielded a list of DEMs of which were upregulated and were downregulated Fig 1C Functional enrichment analysis of overlapped DEGs and target genes of DEMs To assess the biological roles of these DEGs and target genes of DEMs KEGG and GO enrichment analyses were performed The top enriched terms for each analysis were compiled in Fig KEGG pathway enrichment analysis indicated that the DEGs and target genes of DEMs were mainly enriched in osteoclast differentiation complement and coagulation cascades Staphylococcus aureus infection and pertussis Fig 2A GO analysis in the category biological process suggested these DEGs and target genes of DEMs were primarily enriched in ˜cell adhesion™ ˜response to drugs™ and ˜extracellular matrix organization™ Fig 2B GO analysis in the category cellular component suggested that the DEGs and target genes of DEMs were mainly enriched in ˜plasma membrane™ ˜extracellular exosome™ and ˜extracellular localization™ Fig 2C In the category molecular function the DEGs and target genes of DEMs were mostly enriched in ˜protein binding™ ˜identical protein binding™ and ˜calcium ion binding™ Fig 2D PPI network construction and analysis of modules The overlapping DEGs which were shared between the datasets GSE18842 and GSE32863 indicated a distinct set of interactions and networks PPI pairs with a combined score ‰¥ were used to generate the network A PPI network was constructed using of the DEGs and the resultant network had nodes and edges Fig 3A There were upregulated and downregulated genes among the DEGs A total of nodes had a degree of and were designated as hub genes including interleukin Jun protooncogene JUN 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCFigure Functional enrichment analysis of overlapped DEGs and target genes of DEMs A Biological process analysis in the GO database B Cellular component analysis in the GO database C Molecular function analysis in the GO database D Pathway enrichment analysis in the KEGG database The horizontal axis represents the ratio of genes and the vertical axis represents different terms in functional enrichment analysis hsa Homo sapiens DEGs differentially expressed genes DEMs differentially expressed microRNAs GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes ECM extracellular matrix neg negativeubiquitin E2 ligase UBE2C cell division cycle protein CDC20 DNA topoisomerase IIα TOP2A aurora kinase A AURKA AURKB cyclin B2 CCNB2 kinesin family member 20A FBJ osteosarcoma oncogene and maternal embryonic leucine zipper kinase MELK The topology parameters of these hub genes in the PPI network are presented in Table I Furthermore a subnetwork containing nodes and edges was extracted from the global PPI network Fig 3B The results of the KEGG and GO analyses for the genes in the subnetwork are presented in Table II The most significantly enriched terms in this network were ˜cell cycle division™ and ˜DNA replication™ associated with cancer confirming the relevance of the present analysis to NSCLC progression and prognosisSurvival analysis of hub genes Survival analysis was performed for the hub genes based on TCGA data Increased expression of of the hub genes UBE2C CDC20 TOP2A AURKA AURKB CCNB2 and MELK was significantly associated with poorer overall survival OS in patients with LUAD Fig miRNA‘gene network Using the miRWalk application putative DEM targets as established by the TargetScan and miRDB databases were identified A total of putative target mRNAs overlapped with the DEG dataset yielding miRNAgene pairs based on DEMs and the DEGs that were putative targets These were used to construct an overlapping regulatory network Fig A total of upregulated DEMs were predicted to downregulate DEGs whereas decreased expression of DEMs was predicted to be associated with increased expression of DEGs TCGAbased survival analysis suggested that none of the hub genes were associated with OS in patients with NSCLC However specifically among patients with LUAD those with elevated expression of slit guidance ligand SLIT3 phosphoglucomutase PGM5 endomucin EMCN cysteine dioxygenase type CDO1 dihydropyrimidinaselike DPYSL2 miR‘130b and miR‘ had a significantly higher OS compared with patients who had low expression of these genes By contrast elevated miR130b and miR127 expression was associated with poorer OS in patients with LUAD Fig Furthermore the differences of the DEGs and DEMs in miRNAgene networks of NSCLCs with or without KRAS mutation were examined The results suggested that the expression levels of TOP2A P00357 Fig 7A AURKA P00409 Fig 7B and MELK P00190 Fig 7C were significantly lower in KRAS mutation groups compared to KRAS wildtype groups Fig 0cEXPERIMENTAL AND THERAPEUTIC MEDICINE Table I Topology parameters of the genes with a degree ‰¥ in the protein‘protein interaction networkGene IL6 JUN UBE2C CDC20 TOP2A AURKA AURKB CCNB2 KIF20A FOS MELK IL6 interleukin JUN Jun protooncogene UBE2C ubiquitin E2 ligase CDC20 cell division cycle TOP2A DNA topoisomerase Iiα AURKA aurora kinase A CCNB2 cyclin B2 KIF20A kinesin family member 20A FOS FBJ osteosarcoma oncogene MELK maternal embryonic leucine zipper kinaseBetweenness Closeness 974x104 919x104 801x104 715x104 767x104 694x104 717x104 704x104 720x104 872x104 697x104 Stress Degree DownDownUpUpUpUpUpUpUpDownUpMCODE_Score Regulation Figure PPI network and subnetwork A Global PPI network and B the core subnetwork extracted from the global network Red represents upregulation and green represents downregulation The bigger the node the higher the degree PPI proteinprotein interactionDiscussionCancer is a genetic disease wherein cumulative mutations drive the multistep progression towards oncogenesis eventually culminating in unrestrained cancer growth NSCLC remains one of the most common and deadliest forms of cancer making the elucidation of the molecular mechanisms governing this disease paramount In the present study DEMs and DEGs associated with NSCLC were identified via a bioinformatics analysis yielding DEGs and DEMs 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCMCM GTTP MCM CDC GTTP CDC AKRUANEF BNCCMCM CDCMCM x x x noitacilper AND sisoiem etycoO elcyc lleC ash ash ashseneG eulavP oitar eneG syawhtapGGEK A mreTkrowtenbus eht ni seneg eht rof sisylana tnemhcirne OGdna yawhtapGGEK II elbaTseneG eulavP oitar eneG PByrogetac ni sisylana OG B mreTMPSA CEBU BKRUA GTTP CDCGTTP CDC AKRUA AKRUA CRBI FPNEC CRBKLEMI FPNEC XPT AKRUA CRBIKEN BNCC XPT KEN XPT KEN BNCCACDCBNCC x x x noisivid raelcun citotim fo noitisnart MG noisivid llec OGOGOGelcyc llec citotimNEF MCM MCM AAKI CDCCEBU BKRUA GTTP CDC SNGIAKRUABKRUA CDC CRBI FPNECCEBU BKRUA GTTP CDC ACDCAKRUAAPOT MCM MCMMCM MCM CDCMPSA BKRUA AKRUA SMYT x x x x x x x noisehoc ditamorhc retsis noitanitiuqibu nietorp OGOGssecorp cilobatac tnednepedxelpmoc gnitomorpesahpana noitacilper AND OGOGnietorp tnednepednitiuqibu ni ssecorpdevlovni cilobatac noitazinagro eldnips fo noitisnart SG OGOG gnidniwnuAND OGnoitacilper ANDni devlovnielcyc llec citotimAFIKAFIK BKRUA CDC AKRUA AFIK MPSA BKRUA AKRUA CRBI FPNEC PASUN XPT CRPCRBI FPNEC KEN CRP ACDC BKRUA CDC AKRUA CRBI NEF APOT BNCC ACDC FPNECCDC AAKI XPT CRP SMYT MCM CEBU SNGIMCM x x x m ydobdimsalpoelcun eldnips OGOGOG seneG eulavP oitar eneG CCyrogetac ni sisylana OG C mreT\x0cEXPERIMENTAL AND THERAPEUTIC MEDICINE CDCMPSA AKRUA CRBI APOT BNCC FPNECACDC PASUN AAKI XPT KEN CRPCDC SMYT MCM MCM BKRUA BKRUA AKRUA CRBI PASUNCDC FPNEC XPT KENBKRUA AKRUA CRBI PIRT NEF AFIKCDC AKRUA CRBI PASUN XPT KENMCM AKRUA XPT CRP BNCCAKRUA FPNEC KEN BNCC CDC SNGIGTTPKLEM ACDC CRPCRP x x x x x x x xelpmoc regnessap emosomorhc noteleksotyc elubutorcim elubutorcim eldnips elop eldnips elubutorcim emosortnec OGOGOGOGOGOG suelcun OGseneG eulavP oitar eneG CCyrogetac ni sisylana OG C deunitnoC II elbaT mreTPIRT AFIK NEF KLEMAPOT BNCC ACDC CDC MCMCEBU MCMBKRUA AFIKGTTP PIRT KLEM CDC AKRUA APOTCRBI MCMFPNEC CEBU BKRUA MCM AKRUA XPT PASUN AAKI XPT KEN CRP KENSNGIAFIK CRBI PASUN CRPAPOT MCM KLEM BKRUA AKRUA CDCCDC SNGCRBIIKENBKRUA AKRUAKLEM AFIKBKRUA AKRUA AKRUA XPT KENCRPMCM CDC x x x x x x x x x x ytivitca esanik eninoerhtenires gnidnib esanik nietorpnietorp ytivitca esanik gnidnib enires enotsih elubutorcim ytivitca esacilehAND ™™ gnidnib ytivitca esanik nietorpnigiro noitacilper AND gnidnib emyzne OGOGOGOGOGOGOGOG gnidnib nietorp gnidnibPTA OGOG emosomorhcinimortnec FPNEC elcyc noisivid sisenikotyc fo Enitiuqibu CEBU rotaluger nietorp CRP esatehtnys etalydimyht SMYT αiI esaremosiopot AND rotcaretni rotpecer enomroh dioryht APOTPIRT tinubus xelpmoc SNGI A rebmemyli maf nisenik AFIK esanik reppiz enicuel cinoyrbme lanretam nietorp detaicossa eldnips dna raloelcun rotcaf noitaelcun elubutorcimXPT detaicossa elcyc noisivid MCM llec CDC noitarapes ditamorhc retsis fo tnenopmoc ralullec CC llec ACDC selubutorcim eldnips rof rotcaf ylbmessa MPSA B esanik XPT esanikdetaler rotaluger GTTPGTTPAMNI KEN tnenopmoc A esanik arorua xelpmoc ecnanetniam emosomorhcinimAKRUA esaelcunodne cfi arorua ssecorp lacigoloib PB noitcnuf ralucelom FM semoneGdna seneG fo aidepolcycnEotoyK MCM gniniatnoc taeper PAI larivolucab CRBI iceps‘erutcurts pafl NEF tnenopmoc xelpmoc ecnanetniamGGEK elcyc ygolotnO eneG OGnoisivid llec CDC sneipas Bnilcyc omoHBNCC ash KLEMFnietorp esagil erem PASUNBKRUA SNGIseneG eulavP oitar eneG FMyrogetac ni sisylana OG D mreT 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCFigure KaplanMeier survival analysis of the hub genes in the proteinprotein interaction network The KaplanMeier survival analysis based on the expression of A UBE2C B CDC20 C TOP2A D AURKA E CCNB2 F MELK and G AURKB The horizontal axis represents overall survival months and the vertical axis represents the percentage of survival The dotted line indicates the upper and lower boundaries of the confidence interval UBE2C ubiquitin E2 ligase CDC20 cell division cycle TOP2A DNA topoisomerase IIα AURKA aurora kinase A CCNB2 cyclin B2 MELK maternal embryonic leucine zipper kinase HR hazard ratio TPM expressionbased on overlapping hits in the GSE18842 GSE32863 and GSE29250 datasets These hits included upregulated and downregulated genes as well as upregulated and downregulated miRNAs Through functional enrichment analyses it was determined that these DEGs were primarily associated with processes including ˜osteoclast differentiation™ ˜complement and coagulation cascades™ ˜cell adhesion drug responses™ ˜plasma membrane™ ˜extracellular exosome™ and ˜protein binding™ In addition a DEG PPI network was generated and a significant subnetwork module was identified that contained genes associated with the cell cycle DNA replication and oocyte meiosis with the GO terms enrichment for ˜mitotic nuclear division™ ˜cell division™ ˜G2M transition of mitotic cell cycle™ ˜spindle™ ˜midbody™ ˜nucleoplasm™ ˜ATP binding™ ˜protein binding™ and ˜microtubule binding™ Cell cycle dysregulation is known to be a key factor linked to tumor development and progression Recent studies indicated that microtubule binding is linked to tumor metastasis and drug resistance Complement activation and coagulation cascade activation are similarly able to promote tumor development as a consequence of their ability to mediate the recruitment of myeloid cells that support tumor growth To summarize the identified DEGs may regulate the proliferation invasion migration and drugresistance of cancer cells through these pathways thus affecting the occurrence and development of NSCLC The investigation of these DEGs may pave a way towards novel targeted therapies for NSCLC\x0cEXPERIMENTAL AND THERAPEUTIC MEDICINE Figure miRNAmRNA regulatory network The triangles represent miRNAs and the circles represent mRNAs Red indicates upregulation and green indicates downregulation miRNA microRNA miR microRNA hsa Homo sapiensBased on the PPI network hub genes with high degrees of interaction degree ‰¥ were extracted A prognostic analysis of these hub genes was performed using the online tool GEPIA The results revealed that patients with LUAD who had upregulation of UBE2C CDC20 TOP2A AURKA AURKB CCNB2 and MELK had a worse prognosis The expression of UBE2C has been previously indicated to be upregulated in lung cancer and the results of the present study suggested that it was associated with poor survival Similar observations have previously been made in ovarian cancer breast cancer and gastric cancer UBE2C is involved in the progression of the cell cycle and transcription and upregulation of UBE2C may induce an enhanced growth and colony formation of tumors as well as decreased autophagy in cancer cells Furthermore UBE2C reduces the sensitivity of cells to common chemotherapy drugs for lung cancer including cisplatin and docetaxel UBE2C may be used as a therapeutic target for NSCLC Another oncogene in several types of tumor and a hub gene identified in the present study was CDC20 CDC20 is an important regulator of the cell cycle and altered expression or functional impairment may induce mitotic arrest to prevent activation of adenomatous polyposis coli and hence increase premature anaphase manifesting as aneuploidy in daughter cells CDC20 was observed to be upregulated at mRNA and protein levels in NSCLC and was significantly correlated with tumor size pleural invasion and histological classification Of note knockdown of CDC20 caused inhibition of growth migration ability and formation of colonies in lung cancer cells as well as cell cycle arrest in G2M phase and induction of apoptosis making this oncogene a potential target molecule to address NSCLC therapy Upregulation of CDC20 has been associated with shorter OS in patients with LUAD but not in patients with LUSC which is consistent with the results of the present study TOP2A encodes for a DNA topoisomerase involved in torsional dynamics during replication and transcription which is also associated with cell proliferation TOP2A has been indicated to be upregulated in numerous types of tumor including breast nasopharyngeal and renal cell carcinomas and is associated with poor prognosis therefore TOP2A has important roles in cancer The ability of NSCLC cells to proliferate and invade tissues is associated with elevated TOP2A expression Several anticancer agents have been developed to target this gene and the development of drug resistance has been associated with mutation of TOP2A 0cWANG DIFFERENTIALLY EXPRESSED mRNAS AND miRNAS IN NSCLCFigure KaplanMeier survival analysis of the hub genes in the miRNAmRNA regulatory network The KaplanMeier survival analysis based on the expression of A SLIT3 B PGM5 C EMCN D CDO1 E DPYSL2 F MIR130B G MIR1181 and H MIR127 The horizontal axis represents overall survival months and the vertical axis represents the percentage of survival The dotted line indicates the upper and lower boundaries of the confidence interval miRNAmiR microRNA SLIT3 slit guidance ligand PGM5 phosphoglucomutase EMCN endomucin CDO1 cysteine dioxygenase type DPYSL2 dihydropyrimidinaselike TPM transcripts per million HR hazard ratioFigure Violin plots indicating the differences in microRNAgene networks with or without KRAS mutation The expression levels of A TOP2A B AURKA and C MELK were significantly lower in KRAS mutation groups compared to KRAS WT groups P005 TOP2A DNA topoisomerase IIα AURKA aurora kinase A MELK maternal embryonic leucine zipper kinase WT wildtypeAURKA and AURKB are highly conserved serinethreonine kinases the former of which is associated with regulating centrosome duplication and spindle formation and the latter of which is important for regulating chromatin modifications and suppressing cytokinesis NSCLC prognosis is known to be associated with elevated expression of these genes In the present study TCGA dataset analysis suggested that the prognosis of patients with LUAD was associated with AURKA and AURKB which require further clinical trial validation CCNB2 is a cyclin gene that activates cyclindependent kinase to drive the G2M cell cycle transition and inhibition of CCNB2 leads to cell cycle arrest It has been previously confirmed that CCNB2 is upregulated in tissue and serum samples from patients with NSCLC Elevated CCNB2 mRNA levels are known to be closely associated with tumor differentiation grade and histological type and upregulation of CCNB2 at the protein level has been significantly associated with the degree of differentiation tumor size lymph node metastasis distant metastasis and clinical stage Previous studies of NSCLC have suggested that there was no statistically significant correlation between the levels of CCNB2 protein and mRNA in NSCLC The results of the present study indicated that upregulation of CCNB2 mRNA was a poor prognostic biomarker in patients with LUAD while a previous study suggested that the protein levels of CCNB2 may serve as an independent prognostic marker in NSCLC Therefore the role of CCNB2 in NSCLC should be further elucidatedMELK is a serinethreonine kinase that has been indicated to be highly expressed in several human cancer types prostate breast brain colorectal and gastric cancer and glioblastoma multiforme stem cells Elevated expression of MELK is associated with the degree of tumor malignancy and with poor survival in cervical cancer breast cancer and gastric cancer Furthermore the present study suggested that upregulation of MELK is associated with the progression of NSCLC miRNAs regulate a wide array of target mRNAs via 'UTR binding and subsequent translational repression As a 0cEXPERIMENTAL AND THERAPEUTIC MEDICINE result complex miRNAmRNA networks may govern a wide range of biological pathways making miRNAs critical for the progression of numerous types of cancer In the present study a total of DEMs were identified and miRWalk‘mediated predictive analyses were performed to identify those DEMs that were predicted to interact with DEGs yielding hub miRNAs and associated mRNAs including miR1275p miR1345p miR130b3p miR1181 miR1453p miR1533p CDO1 SLIT3 and PGM5 Survival analyses revealed that dysregulation of miR1275p miR130b3p miR1181 CDO1 SLIT3 PGM5 EMCN and DPYSL2 were significantly associated with the prognosis of patients with LUADmiR127 has previously been indicated to function as either a promoter or suppressor of cancer development depending on the specific context Based on the NSCLC network established in the present study miR127 was among the most prominent regulatory miRNAs suggesting it serves complex regulatory functions in the context of NSCLC In a previous study miR127 expression was indicated to be elevated in LUAD and associated with poor prognosis consistent with the results of the present study High levels of miR127 induce epithelialtomesenchymal transition rendering tumor cells with stem celllike properties and propagate tumor resistance to epidermal growth factor receptor inhibitor The aggressiveness of the cancer was associated with a circuit involving miR127 NFκB and tumor necrosis factor αinduced protein which are markers of inflammation miR130b has also been documented in several other types of tumor with upregulation observed in prostate cancer while downregulation was identified in thyroid carcinomas In the present study miR130b3p expression was determined to be significantly increased in NSCLC and associated with poor survival although this was specifically restricted to patients with LUAD in the TCGA dataset warranting further investigation miR134 has been indicated to be differentially regulated in lung cancer and other types of cancer including gastric cancer breast cancer and oral cancer with certain studies reporting increased expression in lung cancer while other studies observed that it was downregulated miR134 may function to either promote or suppress tumor progression highlighting complex mechanisms warranting further investigationmiR1181 has been observed to be downregulated in nasopharyngeal carcinoma ovaria
2
" immune checkpoint inhibitors that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have improved outcomes for many cancer subtypes but do exhibit toxicity in the form of immune related adverse eventsobjective the aim of this study was to investigate the emerging toxicities of pd1 and pd l1 inhibitors including acute or reactivation of tuberculosis tb and atypical mycobacterial infection amimethods this study was completed as a retrospective review using the us food and drug administration adverse events reporting system faers for incidence of tb and ami due to pd1 and pd l1 inhibitors compared with other fda food and drug administration approved drugs the statistical methods included disproportionality signal analysis using the reporting or ror to compare cases the wald ci was reported to assess the precision of the rorresults out of the adverse events aes reported to faers for all drugs between january and march aes were due to the five fda approved pd1pd l1 inhibitors seventy two cases of tb were due to pd1pd l1 inhibitors specifically cases due to nivolumab due to pembrolizumab due to atezolizumab and due to durvalumab there were cases of ami due to nivolumab due to pembrolizumab and each due to durvalumab and atezolizumab avelumab was not attributed to any ae of tb or ami from analysis of the faers database the calculated ror for tb due to pd1pd l1 inhibitors was ci to p00001 and for ami was ci to p00001 pd1pd l1 inhibitors used in the treatment of cancer subtypes is associated with increased tb and ami risk although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of the risksintroductionimmune checkpoint inhibitors icis that block programmed cell death1 pd1 and programmed cell death ligand1 pd l1 have transformed care for many cancer subtypes and have improved outcomes for patients with pd l1 overexpression1 through blockade of the pd1pdl1 axis the t lymphocyte mediated response against tumour cells is enhanced resulting in accelerated immune mediated destruction of key questionswhat is already known about this subject –º case reports and case series suggest programmedcell death1programmedcell death ligand1 pd1pd l1 inhibitors are associated with acute tuberculosis tb or reactivation of tbwhat does this study add –º this is the first large systemic effort to quantify the risk of tb due to pd1pd l1 inhibitors through retrospective analysis of faers food and drug administration adverse events reporting system a pharmacovigilance database pd1pd l1 inhibitors were not only associated with increased risk of tb compared with other drugs but atypicalmycobacterial infection as wellhow might this impact on clinical practice –º although this complication is rare clinicians using pd1pd l1 inhibitors should be aware of thiscancer cells however facilitating immune mediated activation is not benign and patients receiving icis are known to exhibit unique toxicities that result in an damage known as immune related adverse events iraes3 the most common iraes with pd1 and pd l1 inhibitors are fatigue pruritus and diarrhoea4 some iraes can be fatal with pneumonitis hepatitis neurotoxicity and most commonly myocarditis reported5 while counterintuitive when the mechanism of action is considered an emerging and increasingly reported toxicity of pd1 and pd l1 inhibitors is acute tuberculosis tb and reactivation of tb6 the first case of tb due to the pd1 inhibitor was described in a patient with relapsed hodgkin™s lymphoma who developed pulmonary tb following treatment with pembrolizumab7 since then there have been other case reports of tb following initiation of pd1 or pd l1 inhibitors that make the development of tb a relevant concern8“ in a preclinical mouse study pd1 deficient mice were found to be highly susceptible to tb with reduced survival compared with wild type mice12 however anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable adverse events of tb and ami due to pd1pdl1 inhibitors from january to march in faerstotal aes due to all drugs in faerstotal aes due to pd1pdl1 inhibitorstotal aes due to pd1 inhibitorstotal aes of tb in faerstotal aes of tb due to pd1pdl1 inhibitorstotal aes of ami in faerstotal aes of ami due to pd1pdl1 inhibitorsror calculation ror for tb due to pd1pdl1 inhibitors versus full database ror for ami due to pd1pdl1 inhibitors versus full database ci to ci to aes adverse events ami atypical mycobacterial infection faers food and drug administration adverse events reporting system pd1 programmed cell death1 pd l1 programmed cell death ligand1 ror reporting or tb tuberculosisthere is no current risk estimate describing the potential risk of developing tb or atypical mycobacterial infection ami from pd1 and pd l1 inhibitors in this study we retrospectively reviewed the us food and drug administration adverse events reporting system faers a pharmacovigilance database for the risk of tb and ami due to pd1 and pdl1 inhibitors compared with other fda foodand drug administration approved drugsmethodsthis study is a retrospective analysis that used data queries from the faers pharmacovigilance monitoring database faers is a public database that contains nearly million adverse event ae reports medication error reports and product quality complaints reported by healthcare professionals manufacturers and consumers from around the world since these reports are managed by fda and evaluated by clinical reviewers in the center for drug evaluation and research and the center for biologics evaluation and research date in each event report where applicable include individual case identification numbers for reference the suspected pharmaceutical agent treatment indication adverse reactions nature of the event ie serious outcomes eg hospitalised death other outcomes sex male female or unknown age weight event date initial fda receipt date latest fda receipt date pharmaceutical company reporter eg healthcare professional consumer pharmaceutical company unknown concomitant medications latest manufacturer received date country where the event occurred and manufacturer control number individual names and date of birth are excluded from these liststhe present study involved data queries of the faers database between january and march for aes secondary to pd1 inhibitors namely ˜pembrolizumab™ and ˜nivolumab™ and pd l1 inhibitors namely ˜atezolizumab™ ˜durvalumab™ and ˜avelumab™ in all aes due to above five drugs we then searched for three aes specifically ˜tuberculosis™ ˜pulmonary tuberculosis™ and ˜atypical mycobacterial infection™ tuberculosis and pulmonary tuberculosis were grouped together for analysis all other events that were reported in patients with tb or ami were characterised into subcategories including pulmonary infectious endocrine gastrointestinal hepatobiliary dermatological cardiac haematological neurological vascular infusion related rheumatological and otherstb and ami cases among patients treated with pd1 and pd l1 inhibitors were compared with all reported tb and ami events in the database due to other drugs by conducting a disproportionality signal analysis based on the reporting or ror the ror is a measure of the magnitude of association between an exposure to a pharmaceutical agent and the odds of a specific outcome occurring in the setting of an elevated ror it can be conferred that there is an elevated risk of an adverse event occurring with a specific medication the wald ci was used to assess the precision of the ror when lower limit of ror and ci did not cross ror was considered significant13 the likelihood of association between pd1pd l1 inhibitors and tbami were investigated using two sided χ2 or fisher™s exact tests as warranted all analyses were conducted using sas sas institute inc cary north carolina usa and statistical significance was defined as p005resultsbetween january to march a total of adverse events report cases were generated in faers out of ae there were associated with the approved five pd1pd l1 inhibitors the majority of aes were reported with nivolumab and pembrolizumab at in faers there were reports of tb with any drug of which were reported with pd1pdl1 inhibitors the ror for tb due to pd1pd l1 inhibitors was elevated at ci to p00001 for ami there were reports associated with all drugs of which were due to pd1pd l1 inhibitors the anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0ctable details of tb ae due to pd1pdl1 inhibitorstable continuedopen access serious nivolumab pembrolizumab atezolizumab durvalumab lung cancer gastric cancer head and neck cancer hodgkin™s lymphoma malignant melanoma colon cancer neuroendocrine carcinoma ovarian carcinoma pancreatic carcinoma plasma cell myeloma renal cell carcinoma transitional cell carcinoma unknowntotal number of tb aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter year initial report received region of origin of ae died hospitalised life threatening other outcome asia europe americas africa australia healthcare professional consumer male female “ n54 continuedsuspected drug pd1pdl1 inhibitor pd1pdl1 inhibitor ‰¥ aes adverse events pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisror for ami due to pd1pd l1 inhibitors was elevated at ci to p00001 table out of cases of tb due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and due to atezolizumab and durvalumab respectively there were no cases reported with avelumab the most common indication for which pd1pd l1 inhibitor was used was lung cancer median age of the whole cohort was years eighty per cent of the patients were men and were women out of cases cases had a reported outcome of death the most common region of origin in which tb was reported was asia sixteen cases had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table out of cases of ami due to pd1pd l1 inhibitors were due to nivolumab followed by due to pembrolizumab and each due to durvalumab and atezolizumab no report of ami attributable to avelumab was found the most common reason for use of pd1pd l1 inhibitor was lung cancer median age of the entire cohort was years seventy three per cent of patients were men and were women out of cases patient died the most common region in which ami was reported was asia one case had pd1pd l1 inhibitors plus one of more non checkpoint inhibitor drug listed as a suspect drug leading to ae table patients who had tb due to pd1pd l1 inhibitors also had additional reported pulmonary complications in of cases followed by other infectious complications in of cases similarly patients who had ami attributed to use of pd1pd l1 inhibitors had pulmonary complications in of cases followed by endocrine dermatological and others in of the cases table discussionin this retrospective pharmacovigilance database review pd1pd l1 inhibitors had a statistically significant positive signal with tb and ami with a proportion of these events associated with mortality nivolumab had the highest frequency of reported tb and ami whereas avelumab had no reported events most commonly affected patients were receiving treatment for lung cancer and the most commonly reported country of origin was japananand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstb has a high disease burden worldwide with the highest disease associated mortality of any infectious agent in there were million new cases globally and million reported deaths14 amis are estimated to occur in approximately to per persons with an increasing incidence in developed countries15 there is growing evidence that patients receiving icis can develop tb reaction while on treatment6 to date there are reported cases of tb secondary to icis none of which were attributed cytotoxic t lymphocyte associated protein ctla4 inhibitors median time to diagnosis from ici initiation was months range to months17 the mechanism by which a pd1pd l1 inhibitor results in tb is not clear in a murine study pd1 knockout mice had decreased survival compared with wild type mice following exposure to mycobacterium tuberculosis furthermore pd1 inhibition is needed to prevent cd4 t cells from promoting development of tb18 pd1 inhibition mitigates over production of interferon gamma ifnγ which is important for host resistance to tb19increased risk of tb and ami is also found in patients on tumor necrosis factor tnf alpha inhibitors and janus kinase jak inhibitor ruxolitinib20 patients treated with infliximab a tnf alpha inhibitor were and times more likely to develop tb and ami respectively22 in patients prior to start tnf alpha inhibitors screening for latent tb is recommended20 if the patient is found to have latent tb treatment with isoniazid is recommended as it substantially reduces the risk of developing tb reactivation23 however a recent study suggests that pd1 inhibition induced tb reactivation is actually driven by tnf alpha and use of tnf alpha inhibitor could reverse this complication24 there are currently no recommended screening guidelines for latent tb prior to starting pd1pd l1 inhibitors a single institution study in germany found that of patients had positive test for quantiferon gold tb plus qgt prior to starting icis however none of the patients who had a positive qgt test developed tb while on treatment with icis6 of the cases of tb reported in literature due to icis treatment with icis was stopped in all cases tb treatment was initiated and seven cases had re initiation of icis out of seven who had re initiation of ici five had response to therapy one had progression and in one case response was not available17 as tb reactivation may lead to treatment interruptions or discontinuation standardised recommendations for tb screening in patients with planned ici should be considered with substantiation of results from the current study in prospective studiesthis is the first study using faers to demonstrate the potential risk of developing tb and ami in pd1pd l1 inhibitor treated patients as pd1pd l1 inhibitors use becomes more prevalent on a global scale including regions with an elevated prevalence of latent tb clinicians need to consider the risk benefit and economic impacts of screening for latent tb and treatment initiation if the patient is positive these questions cannot be table details of ami ae due to pd1pdl1 inhibitors serious male female nivolumab pembrolizumab atezolizumab durvalumab lung cancer head and neck cancer malignant melanoma unknowntotal number of ami aespd1pdl1 inhibitor used indication for pd1pdl1 use type of reaction sex median age years range min maxoutcome reporter healthcare professionalyear initial report received region of origin of ae suspected drug died hospitalised life threatening other outcome pd1pdl1 inhibitor pd1pdl1 inhibitor ‰¥ asia europe americas “ n10 aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1answered in this observational signal analysis and future prospective research studies should be conducted if a patient develops tb or ami while on treatment with pd1pd l1 inhibitors permanent discontinuation of therapy should be avoided if there is clear clinical benefit from ici and multidisciplinary discussions regarding treatment delay should be conducted with the treating oncologist and infectious disease specialists a majority anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0copen accesstable other aes grouped into major an systems in patients treated with pd1pdl1 inhibitorstb death events n13tb alive events n59tb totaln72ami death events n1ami alive events n12ami totaln13pulmonaryinfectiousendocrinegastrointestinalhepatobiliarycardiachaematologicaldermatologicalneurologicalvascularinfusion relatedrheumatologicalothers aes adverse events ami atypical mycobacterial infection pd1 programmed cell death1 pd l1 programmed cell death ligand1 tb tuberculosisof patients in whom tb or ami have reported are those with lung cancer it is worth pointing out that especially in patients with lung cancer there is significant difficulty in differentiating immune pneumonitis or radiation pneumonitis true disease progression or infectious causes prospective studies of iraes should include testing for tb or ami in diagnostic work upfrom pseudoprogression this study has limitations this analysis was a retrospective study of reported events in faers and as such baseline characteristics including presence of latent tb was not known moreover the actual incidence of tb or ami due to pd1pd l1 inhibitors cannot be determined because faers reports patients with aes not total number of patients taking the medication furthermore it is likely that not all cases of tb that occur in the clinical setting are reported within faers as such there are similar limitations in ror estimate ae reporting for a drug may be influenced by extent of use publicity and bias25 although the use of disproportionality analysis through pharmacovigilance databases to determine the increased risk of aes secondary to particular drug has been shown in various settings25 it is critical that any hypothesis generated by using pharmacovigilance databases are validated through prospective studiespd1pd l1 inhibitors used in treatment for cancer is associated with increased risk of tb and ami the most common drug in faers attributed to tb and ami is nivolumab in this study lung cancer was the most common indication for which use of pd1pd l1 inhibitor leads to tb or ami although this complication is rare clinicians using icis should be aware of this anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866possibility currently there is no additional data available to support or refute the need to screen patients for latent tb prior to initiation of icis prospective studies are needed to address these questions as well as indications to initiate prophylactic therapytwitter vivek subbiah viveksubbiahcontributors conceptualisation ka gs and spi data collection and analysis ka gs je and spi statistics je first draft preparation ka gs and spi review and final approval all authorsfunding 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 vs and spi coi can be found onlinepatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available in a public open access repository all data relevant to the study are included in the article or uploaded as supplementary information faers used for this study is public databaseopen access this is an open access article 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 any changes made are indicated and the use is non commercial see a0http creativecommons licenses by nc orcid idskartik a0anand http orcid vivek a0subbiah http orcid references coit dg thompson ja albertini mr et a0al cutaneous melanoma version nccn clinical practice guidelines in oncology j natl compr canc netw “ ettinger ds wood de aggarwal c et a0al nccn guidelines insights non small cell lung cancer version j natl compr canc netw “ 0copen access postow ma sidlow r hellmann md immune related adverse events associated with immune checkpoint blockade n engl j med “ wang y zhou s yang f et a0al treatment related adverse events of pd1 and pd l1 inhibitors in clinical trials a systematic review and meta analysis jama oncol “ wang dy salem j e cohen jv et a0al fatal toxic effects associated with immune checkpoint inhibitors a systematic review and meta analysis jama oncol “ langan ea graetz v allerheiligen j et a0al immune checkpoint inhibitors and tuberculosis an old disease in a new context lancet oncol 202021e55“ lee jjx chan a tang t tuberculosis reactivation in a patient receiving anti programmed death1 pd1 inhibitor for relapsed hodgkin's lymphoma acta oncol “ fujita k terashima t mio t anti pd1 antibody treatment and the development of acute pulmonary tuberculosis j thorac oncol “ chu y c fang k c chen h c et a0al pericardial tamponade caused by a hypersensitivity response to tuberculosis reactivation after anti pd1 treatment in a patient with advanced pulmonary adenocarcinoma j thorac oncol 201712e111“ anastasopoulou a ziogas dc samarkos m et a0al reactivation of tuberculosis in cancer patients following administration of immune checkpoint inhibitors current evidence and clinical practice recommendations j immunother cancer picchi h mateus c chouaid c et a0al infectious complications associated with the use of immune checkpoint inhibitors in oncology reactivation of tuberculosis after anti pd1 treatment clin microbiol infect “ lázár molnár e chen b sweeney ka et a0al programmed death1 pd1 deficient mice are extraordinarily sensitive to tuberculosis proc natl acad sci u s a “ rothman kj lanes s sacks st the reporting odds ratio and its advantages over the proportional reporting ratio pharmacoepidemiol drug saf “ anization wh global tuberculosis report cassidy pm hedberg k saulson a et a0al nontuberculous mycobacterial disease prevalence and risk factors a changing epidemiology clin infect dis 200949e124“ prevots dr shaw pa strickland d et a0al nontuberculous mycobacterial lung disease prevalence at four integrated health care delivery systems am j respir crit care med “ zaemes j kim c immune checkpoint inhibitor use and tuberculosis a systematic review of the literature eur j cancer “ barber dl mayer barber kd feng cg et a0al cd4 t cells promote rather than control tuberculosis in the absence of pd1 mediated inhibition j immunol “ sakai s kauffman kd sallin ma et a0al cd4 t cell derived ifnγ plays a minimal role in control of pulmonary mycobacterium tuberculosis infection and must be actively repressed by pd1 to prevent lethal disease plos pathog 201612e1005667 solovic i sester m gomez reino jj et a0al the risk of tuberculosis related to tumour necrosis factor antagonist therapies a tbnet consensus statement eur respir j “ anand k burns ea ensor j et a0al mycobacterial infections with ruxolitinib a retrospective pharmacovigilance review clin lymphoma myeloma leuk “ winthrop kl baxter r liu l et a0al mycobacterial diseases and antitumour necrosis factor therapy in usa ann rheum dis “ gómez reino jj carmona l ángel descalzo m et a0al risk of tuberculosis in patients treated with tumor necrosis factor antagonists due to incomplete prevention of reactivation of latent infection arthritis rheum “ tezera lb bielecka mk ogongo p et a0al anti pd1 immunotherapy leads to tuberculosis reactivation via dysregulation of tnfα elife 20209e52668 anand k ensor j trachtenberg b et a0al osimertinib induced cardiotoxicity a retrospective review of the fda adverse events reporting system faers jacc cardiooncology “ anand k ensor j pingali sr et a0al t cell lymphoma secondary to checkpoint inhibitor therapy j immunother cancer 20208e000104anand a0k et a0al esmo open 20205e000866 101136esmoopen2020000866 0c"
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"Introduction Lung cancer is the leading cause of cancer-related deaths worldwide [1] whereas non-small cell lung cancer (NSCLC) represents the most frequent type of lung cancer [2]. NSCLC accounts for approximately 80% of all lung cancer cases and has a 5-year overall survival rate of less than 15% [3 4]. Approximately 40% of patients diagnosed with NSCLC have unresectable stage III disease or medically inoperable disease [5]. Radiation therapy has been regarded as the main treatment strategy for NSCLC for a long time. However radioresistance is the key issue limiting the effects of radiotherapy [2 6]. It is possibly due to tumor heterogeneity in terms of cell of origin pathology etiology and molecular/genetic pathogenesis [7]. NSCLC cells are often resistant to radiotherapy [8] which in turn induces the local recurrence of NSCLC [9 10]. Therefore the development of novel approaches for the treatment of NSCLC including targeted gene treatment as a radiosensitizer to treat this lethal disease is urgently needed to enhance the survival rate in patients. microRNAs (miRNAs) [11] are a class of short noncoding RNAs that function as a regulation for gene expression via targeting mRNA for degradation or inhibition of translation [12]. miRNAs are new factors implicated in regulating the expression of genes involved in tumorigenic processes such as inflammation cell cycle regulation stress response differentiation apoptosis and invasion and over the past decade they have been found to have key roles in cancers [13“15] including lung cancer [16]. Moreover recent studies have suggested a link between expression of some miRNAs and radiotherapy particularly in lung cancer [17“19]. microRNA-21 (miR-21) is a miRNA which has been reported to be overexpressed in many human malignancies including NSCLC [20“22]. Interestingly miR-21 was found to be upregulated in radiotherapy resistant NSCLC cells relative to radiosensitive counterparts [18]. In addition Wang et al. also reported that comparing with radiotherapy resistant NSCLC patients miR-21 was greatly downregulated in radiotherapy sensitive group [23]. Considering miR-21 as a putative regulator of NSCLC radiotherapy resistance we explore the role of miR-21 in radiotherapy resistance of NSCLC A549 cells and the potential molecular mechanism in the present study. 2. Materials and Methods 2.1. Cell Culture The NSCLC cell line A549 was cultured in Dulbecco's modified Eagle's medium (Invitrogen Carlsbad CA) supplemented with 10% fetal bovine serum 100?U/mL penicillin and 100??g/mL streptomycin. Cell cultures were incubated in a humidified atmosphere of 5% CO2 at 37°C. 2.2. Transfection Anti-miR-21 (5?-UCAACAUCA-GUCUGAUAAGCUA-3?) and the negative control oligonucleotides (NC 5?-CAGUACUUUUG-UGUAGUACAA-3?) were obtained from Ambion Inc. (Austin TX USA). The transfection was performed using LipofectamineTM 2000 (Invitrogen USA) according to the instructions provided by the manufacturer. The transfected cells were resuspended and cultured in regular culture medium for 48?h before analysis. 2.3. Detection of miR-21 by TaqMan Real-Time PCR PCR-based detection of miR-21 was performed by the TaqMan miRNA assays (ABI Forest City CA) as described previously [24 25]. The real-time PCR results recorded as threshold cycle numbers (Ct) were normalized against an internal control (U6 RNA) and then expressed as fold changes [25]. 2.4. Ionizing Radiation 48?h after anti-miR-21 or anti-miR-NC transfection subconfluent cell monolayers were treated with ?-ray ionizing radiation (IR) from a 60Co source (PLA General Hospital Beijing China) at a rate of 2.4?Gy/min. 2.5. Clonogenic Survival Analysis After exposure to various doses of IR cells were trypsinized washed and replated at 200 cells per 10-cm dishes. Cells were grown for 14 days fixed with ethanol and stained with Giemsa to detect colonies. The number of colonies containing at least 50 cells was determined and surviving fractions were calculated. 2.6. MTT Assay Twenty-four hours before IR 200??L cells were seeded to 96-well microtiter plate at 5 — 104 cells/mL. Three days after IR 10??L MTT reagent was added to each well followed by incubation for 4?h at 37°C. The supernatants were aspirated and the reaction was terminated by adding 100??L DMSO. The contents of the plates were mixed for 10?min and the absorbance was read at 490?nm. All experiments were performed three times and the average results were calculated. 2.7. Flow Cytometry Attached cells were harvested at 48?h after IR for apoptosis detection using the annexin V-FITC apoptosis detection kit (Sigma Louis MO). Briefly the cells were washed twice with DPBS and then were resuspended in 1— binding buffer at a concentration of 1 — 106 cells/mL. 5??L of annexin V-FITC conjugate and 10??L of propidium iodide solution were added to 500??L of each cell suspension in a plastic 12?mm — 75?mm test tube followed by incubation at room temperature for 15?min and protection from light. The fluorescence of the cells was determined immediately with a flow cytometer. 10?ng/mL of PI3K activator IGF-1 (Prospec-Tany Rehovot Israel) was used in the apoptosis assay. 2.8. Western Blot Analysis Cells were lysed in lysis buffer (20?mM Tris-HCl pH 7.4 150?mM?NaCl 1% Triton X-100 0.1?mM?EDTA 1?mM?EGTA 2?mM sodium orthovanadate 2?mM?NaF and Complete TM Protease Inhibitor Mix [Roche Applied Science Mannheim Germany]) for 20?min on ice and cleared by centrifugation at 12000?rpm and 4°C. Proteins were resolved on a 10% SDS PAGE gel transferred onto nitrocellulose membranes and blocked with 5% nonfat dry milk in TBST (10?mM Tris-HCl pH 7.5 100?mM?NaCl and 0.05% Tween 20) followed by incubation with a primary antibody [total and anti-phosphorylated-Akt (Ser473) antibody (Cell Signaling Biotechnology Beverly MA USA)]. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Antibody complexes were visualized using an enhanced chemiluminescence-Western blotting detection system (Thermo Fisher Scientific Inc. Rockford IL USA). 2.9. Statistical Analysis Statistical analysis was performed using SPSS 13.0. The results from three independent experiments were presented as the means ± standard deviation. Statistical analyses were done by Student's t-test. P value < 0.05 was considered statistically significant. 3. Results 3.1. miR-21 Expression Was Knocked down in A549 Cells by Anti-miR-21 Transfection To confirm knockdown efficiency of anti-miR-21 transfection the relative of miR-21 expression level was detected by real-time quantitative RT-PCR. Compared with anti-miR-NC-transfected A549 cells the level of miR-21 expression in anti-miR-21-transfected cells was significantly decreased by about 64% (). 3.2. Downregulation of miR-21 Inhibited Survival Capacity of A549 Cells after IR To assess whether miR-21 downregulation could sensitize NSCLC A549 cells to IR the A549 cells transfected with either anti-miR-NC or anti-miR-21 were irradiated and their response was analysed. In clonogenic survival analysis we observed the expected decreased survival capacity of A549 cells transfected with anti-miR-21 14 days after IR (). Forty-eight hours after transfection A549 cells were treated with various doses of IR (0246 or 8?Gy) and the survival fractions upon IR were detected. As shown in after IR at 46 or 8?Gy the survival fraction of A549 cells in anti-miR-21-transfected group (0.61 ± 0.06 0.43 ± 0.08 and 0.27 ± 0.07 resp.) was significantly lower than that in anti-miR-NC-transfected group (0.83 ± 0.08 0.76 ± 0.11 and 0.65 ± 0.10 resp.) indicating that downregulation of miR-21 could significantly enhance the sensitivity of A549 cells to IR. 3.3. Downregulation of miR-21 Suppressed Proliferation of A549 Cells after IR To confirm the increased IR sensitivity of A549 cells the effect of miR-21 on cell proliferation was further analysed at 72?h after IR (). Downregulation of miR-21 expression was found to reduce cell proliferation as demonstrated by the decreased proliferation index of cells transfected with anti-miR-21 compared with anti-miR-NC (75.6 ± 18.96% versus 100% P < 0.05). Importantly a more pronounced growth inhibition of A549 cells was found when miR-21 was knocked down in combination with IR. This inhibition of cell growth in the combined treatment (anti-miR-21 + IR) was found to be significantly higher compared with that in the sole IR treatment group (proliferation index: 36.1 ± 8.48% versus 73.2 ± 21.37% P < 0.05 ). This indicates that knockdown of miR-21 sensitizes radioresistant NSCLC A549 cells to radiation. 3.4. Downregulation of miR-21 Enhanced Apoptosis of A549 Cells Induced by IR We next explored the role of miR-21 in the apoptosis of NSCLC A549 cells induced by IR. Anti-miR-21 or anti-miR-NC was transfected into A549 cells and was exposed (or sham exposed) to 8?Gy of IR. As shown in Figure 4 the percentage of apoptosis cells in miR-21 knockdown group (anti-miR-21) was significantly higher than that of negative control group (anti-miRNA-NC) at the dose 8?Gy (61.5 ± 15.62 versus 21.2 ± 5.35 P < 0.05) indicating that miR-21 knockdown may enhance radiosensitivity of A549 cells by promoting apoptosis and thus confirm a role for miR-21 in the regulation of radiotherapy response of NSCLC. 3.5. Downregulation of miR-21 Inactivated PI3K/Akt Signaling Pathway Induced by IR Because the PI3K/Akt signaling pathway is associated with apoptosis we subsequently examined the potential effects of miR-21 on the activation of PI3K/Akt pathways by IR to explore the potential molecular mechanisms. The activation of PI3K/Akt signaling pathways was measured by Akt phosphorylation on Ser473. By Western blot we found that the endogenous level of phospho-Akt expression (Ser473) in anti-miR-21-transfected A549 cells was downregulated compared to that in anti-miR-NC-transfected A549 cells after IR (Figure 5). Interestingly phospho-Akt (Ser473) expression was significantly increased in the case of being treated with IGF-1 a PI3K activator in anti-miR-NC-transfected A549 cells and even in anti-miR-21-transfected A549 cells after IR (Figure 5). This suggested that activation of PI3K/Akt signaling pathway by IR in A549 cells was suppressed by knockdown of miR-21 and the suppression was reversed by PI3K activator IGF-1. 3.6. miR-21 Knockdown Caused Promotion on Apoptosis Induced by IR Was Mediated by PI3K/Akt Signaling Pathway To further confirm the molecular mechanisms of radiosensitization by miR-21 knockdown in NSCLC A549 cells we next treated the cells with or without PI3K activator IGF-1 and then examined the effects of miR-21 downregulation on cell apoptosis induced by IR. As shown in Figure 6 without IGF-1 treatment the cell apoptosis induced by IR was significantly increased in anti-miR-21-transfected A549 cells (61.5 ± 15.62%) compared with that in anti-miR-NC-transfected A549 cells (21.2 ± 5.35% P < 0.05). However after activation of PI3K/Akt signaling pathway the cell apoptosis induced by IR was inhibited in either anti-miR-21-transfected or anti-miR-NC-transfected A549 cells. The percentage of cell apoptosis was not significantly different between these two groups (18.1 ± 5.55% versus 18.3 ± 5.15% P > 0.05). These data showed that in the condition of PI3K/Akt activation knockdown of miR-21 did not promote the apoptosis of A549 cells induced by IR suggesting that PI3K/Akt signaling pathway was the downstream target of miR-21 and the promotive effects of miR-21 knockdown on apoptosis induced by IR were mediated by PI3K/Akt signaling pathway. 4. Discussion It is well known that the acquisition of resistance to radiotherapy which greatly increases patient morbidity and mortality is a significant problem in the treatment of NSCLC. Effective treatment which can sensitize the radioresistant NSCLC to radiotherapy is always being sought. Recently some miRNAs were found to be related to radioresistance. Among them miR-21 is reported to play a role in radioresistance of cancer including glioblastoma [26 27] breast cancer [28] and rectal cancer [29]. But up to now few researches have studied the correlations between miR-21 expression and radiotherapy sensitivity of NSCLC. Liu et al. reported that miR-21 expression promotes radioresistance in NSCLC but the related molecular mechanisms were not revealed [30]. The roles of miR-21 in the radiotherapy response of NSCLC are not fully understood and remain to be elucidated. Thus in the current study we investigated whether miR-21 could affect the radiosensitivity of NSCLC A549 cells and found that downregulation of miR-21 significantly enhanced the sensitivity of A549 cells to radiotherapy through inhibition of PI3K/Akt signaling pathway. Our data showed that following the transfection of anti-miR-21 into A549 cells the inhibition of survival fraction caused by various doses of IR was enhanced compared with radiotherapy alone. This result suggests that miR-21 is closely associated with the therapeutic efficiency of IR on radioresistant A549 cells and downregulation of miR-21 may sensitize A549 cells to IR. It is reported that miR-21 could stimulate growth in NSCLC [30 31]. Accordingly we also found that the proliferation of A549 cells was inhibited after miR-21 knockdown. Moreover the inhibition of cell proliferation induced by combination of miR-21 knockdown and IR was more pronounced compared with either miR-21 knockdown or IR treatment indicating that miR-21 knockdown plays a crucial role in the combined inhibition of cell proliferation and silencing miR-21 may increase the sensitivity of A549 cells to IR. Cell apoptosis induced by IR is one of the most important effects of tumor radiotherapy. Furthermore miR-21 is reported to be an antiapoptotic factor in lung cancer [32 33]. So we hypothesized that it is possible that miR-21 could affect the apoptosis of NSCLC induced by IR. Our current results demonstrated that miR-21 knockdown promoted apoptosis of A549 cells induced by IR indicating that the expression of miR-21 could affect radiosensitivity of NSCLC cells which might be associated with inhibition of apoptosis. This is also in agreement with the previous report [30]. To explore the potential molecular mechanisms of radiosensitization by miR-21 knockdown in NSCLC A549 cells we focused on analysis of PI3K/Akt signaling pathway because the influence of PI3K/Akt signaling pathway on IR-induced apoptotic propensity is well documented [34 35]. We examined whether downregulation of miR-21 could affect Akt phosphorylation at Ser473 and/or its total expression and found that miR-21 knockdown suppressed the activation of PI3K/Akt signaling pathway by IR in A549 cells. In addition the apoptosis induced by IR was enhanced in A549 cells after miR-21 knockdown. This data indicates that the stimulative effects of miR-21 knockdown on A549 cell apoptosis induced by IR are related to the inactivation of PI3K/Akt signaling pathway. Furthermore with the treatment of PI3K activator IGF-1 we found that the apoptosis of A549 cells induced by IR was not promoted even if miR-21 was downregulated. Our results suggest that the promotive effects of miR-21 knockdown on A549 cell apoptosis induced by IR depend on the inactivation of PI3K/Akt signaling pathway."
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in china esophageal cancer ec is the fourth most frequently diagnosed form of malignant tumor inmales and the fifth most commonly diagnosed form in females approximately and casesoccurred in respectively the incidence of ec in eastern asia is in the top five worldwide including china esophageal squamous cell carcinoma escc is a major histological subtype accountingfor of all ec cases the complex interaction of economical and environmental conditions with individual™s hereditary factors may lead to ec development the etiology and development of ec is notfully understood despite many investigations have payed close attention to the importance of immunity recently it was hypothesized that some important variants in immunerelated genes may influencethe susceptibility of esccreceived november revised july accepted july accepted manuscript online august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895interleukin4 il4 coded by the il4 gene is an important regulator of the inflammation pathways il4 apleiotropic cytokine may be correlated with survival and growth of lymphocytes il4 is produced by mast cellprecursors and by the tcell thymocyte populations it is important for bcell activation proliferation and differentiation it is reported that il4 is necessary for producing immunoglobulin e and implicated in immune diseasesin the process of innate immune responses il4 may activate m2 macrophage and then play a specific role it hasantiinflammatory effect which is relevant to the development of escc recently a number of studies have focusedon the relationship of il4 with cancer development il4 single nucleotide polymorphisms snps have alsobeen explored for an association with susceptibility to cancer [“] the rs2070874 tc located in the 5cid3utrregion of the il4 gene is an important snp in cancer development some metaanalyses have indicated that il4rs2070874 may be associated with cancer development in asian populations [“] kim reported that il4rs2070874 might affect the role of aspirin in regulating il4 expression rs2243263 gc polymorphism is anintron snp of il4 gene this intron snp might play a role in splicing although the exact role of this intron snpis unknown the associations of il4 rs2243263 gc snp with the human disease have been explored a previousstudy suggested that il4 rs2243263 was associated with the reverse seroconversion of hepatitis b virus hbv this snp was also studied for the relationship of the susceptibility to cancer a previous report investigated the correlation of the il4 rs2243263 locus with colorectal cancer although in this study a null association was identifiedhowever lan in a large simple size study found that the il4 rs2243263 gc snp might increase the susceptibility to nonhodgkin lymphoma currently the associations of il4 the rs2070874 tc and rs2243263 gcpolymorphisms with escc development are unknownthe il10 gene is located in chromosome 1q322 il10 another immune regulator serves as an inhibitor of dendritic cells and macrophages and inhibits the production of many inflammatory cytokines eg tumor necrosis factorα il1 il6 il12 and others il10 is a vital antiinflammatory regulator after il10 combineswith its receptor il10r signal transducer and activator of transcription is triggered which plays a vital role inantiapoptosis and proliferation an investigation found that the upregulated mrna expression of the il10gene and higher serum levels of il10 were found among subjects who carried the rs1800896 gallele thers1800872 snp a promotor variant could influence the level of il10 protein some investigations have suggested that the il10 rs1800896 ag ˆ’ and rs1800872 ac ˆ’ variants may influence thesusceptibility to escc of late a metaanalysis indicated that these il10 snps increased the risk of ec however in this earlier metaanalysis the sample size was very limited ec patients and controls includedthe association of the il10 rs1800896 ag and rs1800872 ac polymorphisms with ec development should befurther studiedherpesvirus entry mediator hvem also known as tnfrsf14 plays a major role in the immune response[“] hvem has been found to be expressed in lymphoid cells as well as in other cells a previous study suggestedthat the hvemb and tlymphocyte attenuatorlymphotoxincd160 network in immune reaction to infection andinflammation could play a bidirectional regulatory role several investigations have focused on the role of hvemin cancer survival [“] zhu reported that higher expression of hvem may promote apoptosis and herald agood prognosis for bladder cancer patients additionally a previous study has indicated that hvem is implicatedin the development of breast cancer bc a snp in the hvem gene the g to a of rs2234167 in the exon regionwas found to influence the development of bc however the association of hvem rs2234167 ga snp withthe expression of hvem is unknown recently migita found that hvem is critical for both tumor survival andthe escape of the host immune system in escc cases thus it could be a useful target for escc therapy to dateinvestigation has not been performed to identify a relationship of the hvem rs2234167 ga polymorphism withescc susceptibilitytherefore in this investigation the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 andrs1800872 polymorphisms were selected and investigated for their effect on escc development in a chinese hanpopulationmaterials and methodssubjectsour caseˆ’control study was performed in fujian union hospital fuzhou china and the no1 people™s hospital of zhenjiang city zhenjiang china this investigation was approved by jiangsu university registration idk20160036y and fujian medical university registration id 2016zqn25 participants were recruited betweenfebruary and april our study included escc cases and controls these escc patients werehistopathologically confirmed and were from to years old controls were cancerfree individuals from to the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895 years old the controls were not related to any escc case using a prestructured questionnaire we collectedepidemiological data from participants the escc patients and normal controls signed consent formsdna extraction and genotyping of hvem rs2234167 il4 rs2070874 andrs2243263 and il10 rs1800896 and rs1800872 lociwe collected a blood sample ml from each participant dna was extracted carefully as described in a previousstudy using an snpscan„¢ assay genesky biotechologies inc shanghai china we determined the genotypesof hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 polymorphisms to confirm the accuracy of genotyping samples were selected and retested the genotypes of hvem rs2234167 il4rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 loci were reanalyzed by another technician thegenotypes of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 snps wereunchangedstatistical analysisthe difference in alcohol consumption body mass index bmi gender cigarette use and age were tested by using χ2 test mean age was calculated by using a student™s t test we used a chisquare test χ2 or fisher™s exact testto determine whether the frequencies of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 variants in escc cases and controls were different a multivariate logistic regression analysis methodwas used to calculate the crude and adjusted odds ratios ors and confidence intervals cis sas softwarepackage sas institute inc cary nc usa the relationship of hvem rs2234167 il4 rs2070874 and rs2243263and il10 rs1800896 and rs1800872 polymorphisms with escc development was determined by ors and cisthe statistical significance of all analyses was p005 twosided an internetbased hardy“weinberg equilibriumhwe test httpihggsfdecgibinhwhwa1pl was also harnessed to assess whether the distribution of hvemrs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes could represent the included populationresultsbaseline characteristicsin total escc cases and controls were recruited table of these escc cases were females and were males average age was ˆ’ years in the control group there were females and maleswith an average age of ˆ’ years there was no difference in terms of mean age p0413 the categoricalvariables age and gender were wellmatched p005 however the distribution of other categorical variables egtobacco use bmi and drinking status were significantly different all p0001 among escc cases there were with lymphatic metastasis the ajcc version criteria was used to determine the escc stage and escc cases were stage iii and were stage iiiiv after genotyping the participants the association ofhvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes with escc riskwas assessedthe minor allele frequencies mafs of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 loci are shown in table they are similar to the data of chinese population as presented in table the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes in controlsaccorded with hwerelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escctable shows the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypesthe frequencies of il4 rs2070874 tt tc and cc genotypes were and inescc cases and and in controls when the reference was il4 rs2070874 ttgenotype we found the il4 rs2070874 cc genotype significantly decreased the risk of escc p0023 when thereference was il4 rs2070874 tttc genotype the il4 rs2070874 cc genotype also significantly decreased the riskof escc p0028 adjustment for bmi smoking drinking age and gender the decreased susceptibility was alsoidentified cc vs tt p0008 cc vs tttc p0010hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 genotypes are shown in table both crudeand adjusted comparisons indicated that hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 lociwere not associated with the risk of escc table the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0ctable distribution of selected demographic variables and risk factors in escc cases and controlsbioscience reports bsr20193895101042bsr20193895p1variableage yearsage yearssex‰¥malefemaletobacco usenevereveralcohol usenevereverbmi kgm2‰¥lymph node statuspositivenegativetmn stageiiiiiiivgradeg1g2g3cases n721n ˆ’ controls n1208n ˆ’ bold values are statistically significant p005 abbreviation tmn tumorlymph nodemetastasis1twosided χ2 test and student™s t testtable primary information for the included snpsgenotyped polymorphismschromosomeposition regionmaf1 in database 1000g chinese hanpopulatonsmaf in our controls n1208pvalue for hwe2 test in our controls genotyping value1maf2hwehvemrs2234167 gail4 rs2070874tcil4 rs2243263gcil10 rs1800872tgil10 rs1800896tc3cid3utr5cid3utrintron variant5cid3flanking5cid3flankingadditionally a subgroup analysis was conducted by escc stage we identified an association between il4rs2070874 tc snp and the decreased susceptibility of escc in stage iii subgroup cc vs tt p0022 cc vstttc p0025 table however this association could not been identified for other snps the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table the frequencies of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms in different escc subgroupsstage iii patientsstage iiiiv patientsn328n393controls n1208ngenotypehvem rs2234167 gagggaaaa alleleil4 rs2070874 tctttcccc alleleil4 rs2243263 gcgggcccc alleleil10 rs1800872 tgtttgggg alleleil10 rs1800896 tctttcccc alleleoverall casesn721nnnrelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escc in stratified analysesin a stratified analysis the il4 rs2070874 genotypes are listed in table after an adjustment we suggested that il4rs2070874 c allele was a protective factor for escc in five subgroups male subgroup cc vs tt p0028 cc vstttc p0031 ‰¥ years old subgroup cc vs tt p0026 cc vs tttc p0029 never smoking subgroupcc vs tt p0041 cctc vs tt p0013 and tc vs tt p0042 drinking subgroup cc vs tt p0025cc vs tttc p0024 and bmi kgm2 subgroup cc vs tt p0010 cc vs tttc p0012 in othersubgroups no association of l4 rs2070874 with escc risk was found table the il4 rs2243263 gc genotypes in the stratified analysis are listed in table after adjustment we identifiedthat il4 rs2243263 gc polymorphism was a risk factor for escc development in the bmi ‰¥ kgm2 subgroupgc vs gg p0030 and gccc vs gg p0018 table in other stratified analyses adjustment comparisons suggested that hvem rs2234167 and il10 rs1800872 andrs1800896 loci did not confer a risk of escc data not shownassociation of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with lymphatic metastasis in escccasesamong the escc patients patients had lymphatic metastasis as presented in table we found a null association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872 snps with differentlymph node status the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cilcenseccbytheauthorsthisisanopenaccessarticelpublishedbyportlandpressilmitedonbehalfofithebochemcailsocetyianddistributedunderthecreativecommonsattributiontable logistic regression analyses of association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms with risk of esccgenotypehvem rs2234167 gaga vs ggaa vs gggaaa vs ggaa vs gggail4 rs2070874 tctc vs ttcc vs tttccc vs ttcc vs tttcil4 rs2243263 gcgc vs cccc vs gggccc vs ggcc vs gggcil10 rs1800872 tgtg vs ttgg vs ttggtg vs ttgg vs tttgil10 rs1800896 tctc vs ttcc vs tttccc vs ttcc vs tttcpatientsn721vscontrolsoveralln1208crude or cipadjustedor1 ci pstage iii patients n328 vs controlsn1208crude or ciadjustedor1 ci ppstage iiiiv patients n393 vs controlsn1208crude or ciadjustedor1 ci pp “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ 1adjusted for age sex smoking status alcohol use and bmi status bold values are statistically significant p005iiboscencereportsbsrbsr 0cbioscience reports bsr20193895101042bsr20193895table stratified analyses between il4 rs2070874 tc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionil4 rs2070874 tccasecontrol1tttccctttcadjusted or2 ci pcctc cccc vs tcttvariablesexmalefemaleage years‰¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2‰¥ “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p 1for il4 rs2070874 tc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratified factors accordingly] in a logisticregression modelbold values are statistically significant p005association of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with tumor grade of escc casesas presented in table patients had welldifferentiated tumors had moderately differentiated tumors and has poorly differentiated tumors we found an association of the il10 rs1800872 tg snp with a worse differentiation tg vs tt p0048 and ggtg vs tt p0032 table discussionimmunotherapy is altering how we comprehend malignancies and offers new methods to treat them ec is a representative model of immune and inflammationrelated cancer recently some studies indicated that the snps ininflammation and immunerelated genes might influence the risk of ec in this study we explored the role ofimmunerelated gene snps hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872to escc development we observed that il4 rs2070874 tc could decrease a risk to escc even in the stage iiisubgroup however in bmi ‰¥ kgm2 subgroup il4 rs2243263 gc might increase the risk of escc we alsofound an association of the il10 rs1800872 tg snp with a worse differentiationil4 is an important regulator of immune and inflammation pathways some reports have suggested that il4 levelsare higher in untreated escc patients than in controls [“] it is considered that il4 levels may be implicated inthe development of escc the il4 rs2070874 tc polymorphism is a 5cid3utr snp in a highrisk gastric cancergc region a previous study suggested that rs2070874 c allele in the il4 gene might decrease the susceptibilityto gc in a chinese population lu reported that the rs2070874 c allele increased the risk of hcc in amale subgroup however chang and wang found that the il4 rs2070874 polymorphism might notinfluence the susceptibility of cancer in chinese population in this study we included subjects andinvestigated the correlation of this snp to escc susceptibility we found that il4 rs2070874 tc polymorphism the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0ctable stratified analyses between il4 rs2243263 gc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionbioscience reports bsr20193895101042bsr20193895il4 rs2243263 gccasecontrol1gggcccgggcadjusted or2 ci pccgccccc vs gcggvariablesexmalefemaleage‰¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2‰¥ “p “p “p “p p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p “p p “p “p “p 1for il4 rs2243263 gc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratified factors accordingly] in a logisticregression modelbold values are statistically significant p005seemed to be a protective factor for escc development our findings were similar to a previous metaanalysis thatsuggested that the il4 rs2070874 c allele could be associated with a decreased susceptibility of gastrointestinal cancer a functional study indicated that the il4 rs2070874 allele c could promote a higher level of il4 in plasma il4 has an antiinflammatory effect and may decrease the risk of escc by inhibiting the inflammation fitzgerald reported that the il4 rs2070874 allele c could decrease the risk of prostate cancer specific mortality consistent with that report we identified an association between the il4 rs2070874 tc snp and a decreasedsusceptibility to escc in the stage iii subgroup however we did not find an association of il4 rs2070874 tcpolymorphism with lymphatic metastasis this might be due to the limited sample sizes in the future the relationshipof the rs2070874 snp in il4 gene with progress and prognosis should be further exploredrs2243263 gc an intron snp in the il4 gene was studied for the relationship of this snp to some diseasesthis snp might decrease the risk of asthma in the african american children while this relationship was not identified in caucasians hsiao reported that the il4 rs2243263 c allele was a protective factor for hbv surfaceantigen reverse seroconversion in nonhodgkin lymphoma cases undergoing rituximab treatment a previous studyinvestigated the relationship of il4 rs2243263 gc with colon and rectal cancer risk but no association wasfound however in a large simple size study lan found that the il4 rs2243263 gc snp increased the susceptibility to nonhodgkin lymphoma in this study we found that the il4 rs2243263 gc might increase therisk of escc in obese and overweight subjects table it was reported that the il4 level in mothers was inverselylinked to overweight in early childhood and might influence the metabolic profile of childhood in addition thelevel of il4 decreased with antipsychoticinduced weight gain it is suggested that the level of il4 could influence obesity and overweight introns are regulatory sequences that can affect the expression of genes here we foundthat the rs2243263 gc polymorphism a snp in il4 intron region might alter the risk of escc it is presumed the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and lymph node status in escc patientsgenotypepositive n405negative n316crude or cipadjusted or1 ciphvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenn1adjusted for age sex smoking alcohol use and bmi status “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “that the rs2243263 gc polymorphism influences the level of il4 by regulating gene transcription in the future afunctional study should be considered to explore the potential mechanismthe il10 rs1800872 tg is a promotor snp torrespoveda reported that the expression of il10 mrnaand the level of serum il10 were significantly higher in subjects with the il10 rs1800872 t allele a recent studyfound that il10 rs1800872 tg snp promoted the risk of ec a metaanalysis also confirmed this association in our case“control study we did not find the association of il10 rs1800872 tg snp with the developmentof ec even in stratified analyses and reviewing different lymph node status additionally liu reported thatil10 rs1800872 gg genotypes predicted the worse survival of diffuse large bcell lymphoma patients treated withrituximabchop cyclophosphamide doxorubicin vincristine and prednisone in this study we found that the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and grades of esccgenotypeg2g3 n579hvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenng1 n142crude or cipadjusted or1 cip “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “1adjusted for age sex smoking alcohol use and bmi statusbold values are statistically significant p005the il10 rs1800872 g allele was associated with poorly differentiated tumor thus in the future the association ofthe il10 rs1800872 tg snp and the survival of escc cases should be further studiedlimitations in the present study should be acknowledged first in the present study we only included five functional snps and explored the association of the risk to escc second there were other environmental risk factorseg vegetable and fruit intake aspirin and nsaids use and physical exercise which we did not consider for theirinfluence to the development of escc third the number of escc patients was limited and our study may be the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895underpowered in some subgroups fourth in this investigation the protein expression levels of the suspect factors were not measured finally considering the low penetrance of snp the other functional polymorphisms in thehvem il4 and il10 genes should not be ignoredin summary the present study suggests that the il4 rs2070874 tc polymorphism is a protective factor for esccdevelopment while the il4 rs2243263 gc increases a risk to escc in obese and overweight subjects additionallyit is highlighted that the il10 rs1800872 g allele is associated with poorly differentiated tumorcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis work was supported in part by the young and middleaged talent training project of health development planning commission in fujian province [grant number 2016zqn25] the program for new century excellent talents in fujian province university[grant number ncetfj2017b015] and the joint funds for the innovation of science and technology fujian province [grantnumber 2017y9099]author contributionall authors contributed significantly to the present study conceived and designed the experiments wt and mk performed theexperiments sc rc and cl analyzed the data wt and mk contributed reagentsmaterialsanalysis tools mk wrote themanuscript sc and rc other please specify noneacknowledgementswe appreciate all subjects who participated in the present study we wish to thank dr yan liu genesky biotechnologies incshanghai china for technical supportabbreviationsajcc american joint committee on cancer bc breast cancer bmi body mass index ci confidence interval ecesophageal cancer escc esophageal squamous cell carcinoma gc gastric cancer hbv hepatitis b virus hvem herpesvirus entry mediator hwe hardy“weinberg equilibrium il interleukin nsaid nonsteroidal antiinflammatory drug or odds ratio snp single nucleotide polymorphismreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ 103322caac21492 matejcic m and iqbal parker m geneenvironment interactions in esophageal cancer crit rev clin lab sci “1031091040836320151020358 qin jm yang l chen b interaction of methylenetetrahydrofolate reductase c677t cytochrome p4502e1 polymorphism andenvironment factors in esophageal cancer in kazakh population world j gastroenterol “ pmcid pmc2773864103748wjg146986 lin ew karakasheva ta hicks pd the tumor microenvironment in esophageal cancer oncogene “ pmcidpmc5003768 101038onc201634 park r williamson s kasi a immune therapeutics in the treatment of advanced gastric and esophageal cancer anticancer res “ 1021873anticanres12891 nelms k keegan ad zamorano j the il4 receptor signaling mechanisms and biologic functions annu rev immunol “ 101146annurevimmunol171701 rush js and hodgkin pd b cells activated via cd40 and il4 undergo a division burst but require continued stimulation to maintain divisionsurvival and differentiation eur j immunol “101002152141412001043143c1150aidimmu11503e30co2v suzuki a leland p joshi bh targeting of il4 and il13 receptors for cancer therapy cytokine “101016jcyto201505026 francipane mg alea mp lombardo y crucial role of interleukin4 in the survival of colon cancer stem cells cancer res “ 10115800085472can076874 tan n song j yan m association between il4 tagging single nucleotide polymorphisms and the risk of lung cancer in china molgene genom med e00585 pmcid pmc6465665 101002mgg3585 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20193895101042bsr20193895 cardenas dm sanchez ac rosas da preliminary analysis of singlenucleotide polymorphisms in il10 il4 and il4ralpha genesand profile of circulating cytokines in patients with gastric cancer bmc gastroenterol pmcid pmc6288868101186s1287601809139 shamoun l skarstedt m andersson re association study on il4 il4ralpha and il13 genetic polymorphisms in swedish patientswith colorectal cancer clin chim acta “ 101016jcca201809024 jia y xie x shi x associations of common il4 gene polymorphisms with cancer risk a metaanalysis mol med rep “ pmcid pmc5561993 103892mmr20176822 cho ya and kim j association of il4 il13 and il4r polymorphisms with gastrointestinal cancer risk a metaanalysis j epidemiol “ pmcid pmc5394226 101016jje201606002 zhenzhen l xianghua l qingwei w three common polymorphisms in the il4 gene and cancer risk a metaanalysis involving cases and controls tumour biol “ 101007s1327701307618 kim bs park sm uhm tg effect of single nucleotide polymorphisms within the interleukin4 promoter on aspirin intolerance inasthmatics and interleukin4 promoter activity pharmacogenet genomics “ hsiao lt wang hy yang cf human cytokine genetic variants associated with hbsag reverse seroconversion in rituximabtreatednonhodgkin lymphoma patients medicine baltimore e3064 pmcid pmc4839912 101097md0000000000003064 bondurant kl lundgreen a herrick js interleukin genes and associations with colon and rectal cancer risk and overall survival intj cancer “ pmcid pmc3470814 101002ijc27660 lan q wang ss menashe i genetic variation in th1th2 pathway genes and risk of nonhodgkin lymphoma a pooled analysis ofthree populationbased casecontrol studies br j haematol “ pmcid pmc3075370101111j13652141201008424x kwasniak k czarnikkwasniak j maziarz a scientific reports concerning the impact of interleukin interleukin and transforminggrowth factor beta on cancer cells central eur j immunol “ pmcid pmc6745546 105114ceji201876273 d™andrea a asteamezaga m valiante nm interleukin il10 inhibits human lymphocyte interferon gammaproduction bysuppressing natural killer cell stimulatory factoril12 synthesis in accessory cells j exp med “ pmcid pmc2191152101084jem17831041 miteva ld stanilov ns deliysky ts significance of 1082ag polymorphism of il10 gene for progression of colorectal cancer andil10 expression tumour biol “ 101007s1327701425892 pereira apl trugilo kp okuyama ncm il10 c592ca rs1800872 polymorphism is associated with cervical cancer j cancerres clin oncol “ 101007s00432020032560 yang y and fa x role of il10 gene polymorphisms on the susceptibility for esophageal cancer and its association with environmental factorsint j clin exp pathol “ pmcid pmc4583954 sun jm li q gu hy interleukin rs1800872 tg polymorphism was associated with an increased risk of esophageal cancer in achinese population asian pac j cancer prev “ zhao x lu c chu w 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"The principal function of iodine acts on thyroid function but in recent years the role of iodinedeficiency in metabolism has also been gradually revealed We aimed to investigate the current status of iodizedsalt consumption and urinary iodine concentration UIC in an urban Chinese population with type diabetes andto further explore whether UIC was associated with diabetic microvascular complicationsMethods Four thousand five hundred fiftynine subjects with diabetes from communities in downtownShanghai were enrolled in the crosssectional Metal Study in UIC was detected using an inductively coupledplasmamass spectrometer Diabetic kidney disease DKD was defined as urinary albumintocreatinine ratioUACR mgg or estimated glomerular filtration rate mLmin173 m2 Diabetic retinopathy DR wasevaluated by highquality fundus photographs and was remotely read by ophthalmologistResults The median UIC of subjects with diabetes was μgL “ in downtown Shanghai Among allthe subjects consumed noniodized salt and were iodine deficient Iodine deficiency UIC μgLwas associated with an increased odds of DKD OR 95CI “ after adjustment for age sex educationcurrent smokers BMI HbA1c duration of diabetes dyslipidemia thyroidstimulating hormone and free thyroxineNo association was observed between UIC and DR after multivariable adjustmentConclusions A concerning number of subjects with diabetes consumed noniodized salt and suffered from iodinedeficiency in coastal regions of China Low UIC might be a risk factor for DKD which should be further confirmedby longitudinal prospective studiesKeywords Iodized salt Type diabetes Diabetic kidney disease Urinary iodine concentration Epidemiology Correspondence wnj486126com luyingli2008126com Chi Chen and Yi Chen contributed equally to this workInstitute and Department of Endocrinology and Metabolism Shanghai NinthPeople™s Hospital Shanghai JiaoTong University School of MedicineShanghai China 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 Nutrition Metabolism Page of IntroductionType Diabetes Mellitus T2DM has become a seriousglobal health care burden causing microvascular complications which are associated with increased disabilityreduced quality of life and life expectancy [ ] It wasestimated that there were million cases of adult diabetes worldwide in and the number was projectedto increase to million by [] leading to highincidence and prevalence of microvascular complications Approximately one third with T2DM will developdiabetic retinopathy DR and one quarter will developdiabetic kidney disease DKD [] Thus it is critical toidentify and control some novel modifiable risk factorscontributing to microvascular complications in patientswith T2DMIodine is an indispensible micronutrient for the synthesis of thyroid hormones Iodine deficiency ID in early lifeimpairs neurodevelopment and also has many adverse effects throughout various life stages [] Over the past years substantial progress has been achieved in the worldwide effort to eliminate iodine deficiency disorders IDDby salt iodization program However this program is vulnerable and requires a longterm commitment from governments In several countries where ID had been onceeliminated salt iodization programs were discontinuedand ID has now reappeared []The principal function of iodine acts on thyroid function but in recent years the role of ID in metabolismhas also been gradually revealed Analysis of data fromthe National Health and Nutrition Survey NHANES“ found that in US adults low urinary iodineconcentration UIC was associated with dyslipidemia[] and coronary artery disease [] Moreover O S AlAttas [] reported that UIC is markedly decreasedin T2DM and urinary iodine was negatively associatedwith insulin resistance in patients with T2DM Most recently Mingyue Jin [] also found that at a lowerUIC μgL the prevalence of diabetes significantlyincreased relative to an UIC of “ μgL Animalstudies also showed that iodine supplementation couldreduce the blood glucose levels and improve the insulinsensitivity in goats [] However the role of iodine nutrition on diabetic microvascular complications has notbeen studiedThe aim of this study was to investigate the currentstatus of iodized salt consumption and UICs in an urbanChinese population with T2DMand additionallywhether UIC is associated with diabetic microvascularcomplications including DKD and DRMethodsStudy populationThe crosssectional METAL study Environmental Pollutant Exposure and Metabolic Diseases in Shanghaiwwwchictrcn ChiCTR1800017573 was launchedto investigate the association between iodine nutritionand microvascular complications in Chinese adults withdiabetes We recruited study participants from sevencommunities in Huangpu and Pudong new districtShanghai China Huangpu district located in downtownShanghai is the administrative economic and culturalcenter of the metropolitan coastal city [] Pudong newdistrict is the symbol of China™s reform and ingup[] We randomly selected half of patients with diabetesfrom the registration platform in each communityhealthcare center Chinese citizens ‰¥ years old whohad lived in their current area for ‰¥ months were included In August a total of subjects withT2DM who were “ years of age received an examination Participants with missing UIC values n were excluded Finally participants were involvedin the present analysisThe study received ethical approval from the EthicsCommittee of Shanghai Ninth People™s Hospital Shanghai Jiao Tong University School of Medicine All procedures followed were abided by the ethical guidelines ofthe Declaration of Helsinki as reflected in a prioriapproval by the appropriate institutional review committee Informed consent was received from all participantsincluded in the study prior to the data collectionMeasurementsinThe same welltrained and experienced personnelSPECTChina study [“] used a questionnaire to collect information on sociodemographic characteristicseducation medical history family history and lifestylerisk factors Weight and height were measured using abalance beam and a vertical ruler in light clothing andwithout shoes Body mass index BMI was calculated asthe ratio of weight in kilograms divided by height in meters squared Current smoking was defined as havingsmoked at least cigarettes in one™s lifetime and currently smoking cigarettes [] Especially œFor the pastthree years which type of salt was used in your familywas applied to collect information about type of saltThree options for this item were provided only iodized salt only noniodized salt bothBlood samples were drawn between am and am after an overnight fast Blood was refrigerated immediately after phlebotomy and in hours it was centrifugated and the serum was aliquoted and frozen in acentrallaboratory Glycated hemoglobin HbA1c wasmeasured by highperformance liquid chromatographyMQ2000PT Medconn Shanghai China Fastingplasma glucose serum creatininetotalcholesterol high HDL and lowdensity lipoproteinLDL were performed with a Beckman Coulter AU Brea USA Serum thyroidstimulating hormone TSHtriglycerides 0cChen Nutrition Metabolism Page of and free thyroxine FT4 were measured by electrochemiluminescence Roche E601 GermanyMorning fasting spot urine samples collected were refrigerated immediately and frozen at ˆ’ °C in hoursUIC was detected using an inductively coupled plasmamass spectrometer ICPMS No 7700x Agilent Technologies Inc USA The concentrations of urine albuminand creatinine were determined with a Beckman CoulterAU Brea USA using a turbidimetric immunoassayand an enzymatic method respectively Urinary albumintocreatinine ratio UACR was calculated as the urinaryalbumin concentrations divided by the urinary creatinineconcentrations and expressed in mggDR screening was evaluated by mydriatic binocular indirect ophthalmoscopy Topcon TRCNW400 NonMydriatic Retinal Camera Oakland USA Fundus photographs were read by an experienced ophthalmologistspecialized in retinaOutcome definitionDyslipidemia was defined as total cholesterol ‰¥mmolL mgdL triglycerides ‰¥ mmolL mgdL LDL ‰¥ mmolL mgdL HDL mmolL mgdL or selfreported previous diagnosisof hyperlipidemia by physicians according to the modified National Cholesterol Education ProgramAdultTreatment Panel IIIThe estimated glomerular filtration rate eGFR was calculated using the Chronic Kidney Disease EpidemiologyCollaboration CKDEPI equation for œAsian origin []As suggested by American Diabetes Association ADAhigh UACR was defined as UACR ‰¥ mgg reducedeGFR as eGFR mlmin173m2 and DKD as UACR mgg or eGFR mLmin173 m2 []The internationally accepted DR classification by theœGlobal Diabetic Retinopathy Project Group in was applied [] The classification was no retinopathynonproliferative DR intraretinal microaneurysmshemorrhages venous beading prominent microvascularabnormalities and proliferative DR neovascularizationor vitreouspreretinal hemorrhagesStatistical analysisStatistical analysis was run with SPSS IBM Corporation Armonk NY USA General characteristics arepresented as median with the interquartile range IQRfor continuous variables or as proportion for categoricalvariables MannWhitney U test and the KruskalWallistest were used for comparison of two or more groups ofnonnormally distributed data Pearson™s χ2 tests wereperformed to compare categorical variablesThe associations of UIC with elevation of UACR reduction of eGFR DKD and DR were analyzed with logistic regression analyses The results are presented as oddsratio OR and confidence intervals CIs Model was unadjusted Model was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Table General characteristics of study participants by UIC categoriesUrinary iodineAge yrAdequate “ “ “ “ “ “Low “ “ “ “ “ “Women UIC μgLFPG mmolLHbA1c BMI kgm2Duration of diabetes yrCurrent smokers Beyond high school education TSH mIULFT4 pmolLBlood lipidsTotal cholesterol mmolLLDLC mmolLHDLC mmolLTriglycerides mmolLMore than adequate “ “ “ “ “ “ “ “Excessive “ “ “ “ “ “ “ “ “ “ “ “P “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Data are summarized as median interquartile range for continuous variables or as number with proportion for categorical variablesUIC Urinary iodine concentration FPG Fasting plasma glucose HbA1c Glycated hemoglobin BMI Body mass index LDLC Low density lipoprotein cholesterol HDLC High density lipoprotein cholesterol TSH Thyroidstimulating hormone FT4 Free thyroxineUrinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL 0cChen Nutrition Metabolism Page of ResultsGeneral characteristics of the study populationThe general characteristics are presented in Table The mean age of the study population was nearlyone half were women About of the studypopulation were overweight or obese BMI ‰¥ kgm2and were current smokers The percentage of aneducational level beyond high school was and theaverage duration of diabetes was yearsCompared to those with adequate iodine nutritionsubjects with ID were slightly older and were more likelyto be women These subjects also had higher TSH lowerBMI and a lower percentage of current smokers Inaddition subjects with more than adequate and excessiodine nutrition were slightly younger and had higherBMI but comparable TSHUrinary iodine concentration and type of salt intake inthe populationThe distribution of UICs in the study population is presented in Fig The median 25th“75th percentile UICof subjects with diabetes was μgL “ indowntown Shanghai which falls within the range of μgL that WHOUNICEFICCIDD categorize asadequate Urinary iodine measurements indicative of IDUIC μgL were present in of the studypopulation Meanwhile and of the populationshowed more than adequate UIC “ μgL andexcess iodine intake UIC ‰¥ μgL respectivelyThe distribution of type of salt intake is presented inFig As high as of the study population consumed noniodized salt consumed iodized saltand consumed both Logistic regression analysisshowed that compared to those who consumed iodizedsalt subjects consumed noniodized salt were morelikely to be women OR 95CI “ and havea higher educational attainment OR 95CI “ but a comparable age OR 95CI “Association of urinary iodine concentration with elevationof UACR reduction of eGFR and DKDThe association of UIC with elevation of UACR reduction of eGFR and DKD is shown in Table Comparedwith those with adequate iodine nutrition subjects withID had an increased risk of elevation of UACR OR 95CI “ reduction of eGFR OR 95CI“ and DKD OR 95CI “ Adjustment for age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 did not attenuate the association of ID with UACRand DKD However multivariable adjustment weakenedthe association between ID and reduction of eGFR further such that it was no longer significant Meanwhilesubjects with more than adequate and excess iodine nutrition did not have an increased risk of elevation ofUACR reduction of eGFR and DKD after multivariableadjustmentAssociation of urinary iodine concentration with DRTable presents the association of UIC with nonproliferative and proliferative DR Compared with thosewith adequate iodine nutritionthe ORs of nonproliferative and proliferative DR in subjects with morethan adequate iodine nutrition were 95CI “Fig Distribution of UICs in the study population 0cChen Nutrition Metabolism Page of Fig Distribution of type of salt consumed in the study populationthan adequate and 95CI “ respectively Multivariable adjustment weakened the association betweenmoreiodine nutrition and nonproliferative DR further such that the association was nolonger significant There was no significant associationobserved between DR and ID and excessiodinenutritionDiscussionIn this study among over communitydwellingChinese adults with diabetes we found that of thesubjects consumed noniodized salt and had IDIodine deficiency was significantly associated with ahigher prevalence of elevated UACR and DKDindependently of age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 To the best of our knowledge this is the first studyto investigate the current status of iodized salt consumption and iodine nutrition status in a relatively largepopulation with diabetes and further investigate the association between UIC and diabetic microvascularcomplicationsChina was once severely affected by IDD and hence amandatory Universal Salt Iodization USI program wasTable Association of urinary iodine with elevation of UACR and reduction of eGFRUrinary iodineHigh UACRAdequateLowMore than adequateExcessivePrevalenceOdds RatioModel Model Reduced eGFRPrevalence Odds RatioModel Model DKDPrevalence Odds Ratio Ref Ref Ref Ref “ “ “ “ “ “ “ “ “ “ “ “Model Model Ref Ref “ “Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with elevation of UACR reduction of eGFRand DKD P Model was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4High UACR was defined as UACR ‰¥ mgg reduced eGFR as eGFR mlmin173 m2 and DKD as UACR mgg or eGFR mLmin173 m2Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL “ “ “ “ 0cChen Nutrition Metabolism Page of Table Association of urinary iodine with DRUrinary iodineNonproliferative DRAdequatePrevalence Odds RatioModel Model Proliferative DRPrevalence Odds RatioModel Model Ref Ref Ref RefLowMore than adequateExcessive “ “ “ “ “ “ “ “ “ “ “ “P Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with DRModel was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgLintroduced in and was successful in eliminatingIDD However since the prevalence of thyroid diseaseshas markedly increased in recent years some concernsabout the USI have circulated especially among coastalresidents in urban areas [] In the present analysis themedian UIC of residents with diabetes in downtownShanghai has fallen to marginal levels of iodine sufficiency μgL and more surprisingly more thanhalf of the subjects consumed noniodized saltand were iodine deficient Compared with a studyconducted by the Shanghai Municipal Center for DiseaseControl and Prevention CDC in of participants used iodized salt and were iodine deficientat that time [] During “ Zhongyan Shanand her colleagues performed a crosssectional study ineastern and central China and reported that the medianUIC was μgL in schoolaged children and μgLin the total cohort population [] Our previous studyconducted on the general population found that consumed noniodized salt in the urban area of Shanghai in []The present study indicates that an increasing numberof urban residents in downtown Shanghai prefer to usenoniodized salt in recent years and suffer from IDWomen and those with a higher educational level weremore tended to consume noniodized salt WhyChanges in the reported spectrum and growing incidence of thyroid disorders have been linked to the increased iodine intake resulting from USI in the localmedia and international medical literature [] Thosewith high educational attainment were more likely to beconfused by these information and worry about theirthyroid health and call for liberalizing provincial controlof sales of noniodized salt Formerly needed a prescriptionthe sale of noniodized salt has now beenunofficially allowed by some coastal city authorities []In addition T2DM is associated with an increased riskof multiple thyroid disorders such as thyroid nodule[] thyroid cancer [] and autoimmune thyroid diseases [] Since the prevalence of thyroid abnormalitieswere found to be much higher in females than males[] it is reasonable to deduce that these women wouldhave a higher tendency towards consuming noniodizedsalt after diagnosis of thyroid abnormalitiesseafood alone to provide sufficientConsidering that Shanghai is a coastal city local residents believe that they should never suffer from IDDwith high iodineenriched aquatic products consumption However it may not be true to rely on consumption ofiodineActually the environmental levels of iodine in Shanghaiare deficient μgL [] Furthermore based on theresearch initiated by Shanghai Municipal CDC iodizedsalt contributed of the total dietary iodine inShanghai[] Aquatic products which residentsthought to be rich in iodine accounted for only ofthe total dietary iodine []Thus iodized salt is still themain source foriodine supplementation in coastalpopulationsDKD wascommonlydefinedby ADA asœUACR‰¥30mgg or eGFR60 mlmin per m2 []We found that ID was associated with elevated UACRand DKD The mechanism underlying the association ofID with DKD is not yet fully understood Deficiency ofiodine could reduce thyroid hormone production andelevate TSH It has been reported that lower free triiodothyronine and elevated TSH have significant associationwith risk for albuminuria in T2DM [ ] Moreoverinadequate iodine intake is significantly correlated withan increase in oxidative stress [] Recent evidence hasshown thattumorinflammatory cytokinessuch as 0cChen Nutrition Metabolism Page of necrosis factoralpha and interleukin1 play a pivotalrole in the pathogenesis of DKD [] Therefore we suppose the possible mechanism may be via inflammatoryresponseIn view of the evergrowing prevalence of T2DM andDKD all over the world successive intervention in thislarge population can have important impact on publichealth ID unlike most micronutrient deficiencies is notrestricted to people in developing countries with poordiets Since salt iodization is simple effective and inexpensive the best strategy to control ID is addition ofiodine into salt in nearly all countries [] Monitoringiodine situation of people with diabetes is of critical significance and education programs to diabetes especiallywomen with high academic background may also include information of adequate iodine intake in our clinical practice Our study shed light on the possiblebeneficial effect of iodine supplementation in reducingalbuminuria in T2DM which warrants further investigation in welldesigned randomized controlled trialOur study benefited from its welldefined communitybased participants with a relatively large sample sizeSecond regarding the novelty our study is the first toprovide iodine status and linked iodine insufficiency toan increased risk of albuminuria in people with diabetesThird we used ICPMS to detect UIC in the presentanalysis which was considered as the goldstandardmethod [] There were also some limitations weshould acknowledge First no causal relationship couldbe determined due to the crosssectional design of thestudy and thus our findings need to be validated by longitudinal prospective studies Second although UIC isrecommended by the WHOICCIDDUNICEF for evaluation of iodine status at the population level and widelyused in largescale epidemiological studies [ ] thesingle spot urine measurement may not accurately assesslongterm iodine status at the individual level Actuallyinter and intraindividual variability exists in UIC []However the application of a large sample size from to subjects per subgroup may counteract thebias related to the use of only one casual urine sample[] Future followup studies collecting 24h urine specimens twice are needed to replicate the present resultsConclusionA large proportion of diabetic patients in downtownShanghai consumed noniodized salt and had ID IDmay increase the risk of DKD independent of thyroidfunction in diabetic patients Maintaining USI at an appropriate level is indispensable for diabetic patients Cohort and intervention studies as well as basic researchexploring the effect and mechanism of iodine supplementation on renal function are warrantedAbbreviationsT2DM Type Diabetes Mellitus DKD Diabetic kidney disease DR Diabeticretinopathy ID Iodine deficiency UIC Urinary iodine concentrationTSH Thyroidstimulating hormone BMI Body mass index OR Odds ratioCI Confidence interval UACR Urinary albumintocreatinine ratioeGFR Estimated glomerular filtration rateAcknowledgementsThe authors thank Xiaojin Wang and Bingshun Wang from the Departmentof Biostatistics and Shanghai Jiaotong University School of Medicine for dataprocessingAuthors™ contributionsYL and NW designed the study CC YC HZ FX BH WZ YW HWand NW conducted the research CC and YC analyzed the data and wrotethe manuscript CC and YC contributed equally The final manuscript wasread and approved by all authorsFundingThis study was supported by National Natural Science Foundation of China Science and Technology Commission of ShanghaiMunicipality the Fourth Round of ThreeYear Public HealthAction Plan of Shanghai by the Shanghai Municipal Commission of Healthand Family Planning 20164Y0079 Municipal Human Resources DevelopmentProgram for Outstanding Young Talents in Medical and Health Sciences inShanghai 2017YQ053 Fundamental research program funding of NinthPeople™s Hospital affiliated to Shanghai Jiao Tong university School of MedicineJYZZ099 Shanghai Sailing Program 20YF1423500Availability of data and materialsThe datasets during andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Ethics Committee of Shanghai NinthPeople™s Hospital Shanghai Jiao Tong University School of Medicine Writteninformed consent was received from all participantsConsent for publicationNACompeting interestsNo potential conflicts of interest relevant to this were reportedReceived May Accepted August ReferencesKahm K Laxy M Schneider U Rogowski WH Lhachimi SK Holle R Healthcare costs associated with incident complications in patients with type diabetes in Germany Diabetes Care “Chen C Chen Q 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crosssectional study in cities Thyroid “ Chen C Xu H Chen Y Chen Y Li Q Hu J Iodized salt intake and itsassociation with urinary iodine thyroid peroxidase antibodies andthyroglobulin antibodies among urban Chinese Thyroid “Teng X Shan Z Chen Y Lai Y Yu J Shan L More than adequateiodine intake may increase subclinical hypothyroidism and autoimmunethyroiditis a crosssectional study based on two Chinese communities withdifferent iodine intake levels Eur J Endocrinol “ Wu Y Li X Chang S Liu L Zou S Hipgrave DB Variable iodine intakepersists in the context of universal salt iodization in China J Nutr “ Chen Y Zhu C Chen Y Wang N The Association of Thyroid Nodules withmetabolic status A crosssectional SPECTChina study Int J Endocrinol Qi J He P Yao H Song R Ma C Cao M Cancer risk among patientswith type diabetes a realworld study in Shanghai China SarfoKantanka O Sarfo FS Ansah EO Yorke E Akpalu J Nkum BC et alFrequency and determinants of thyroid autoimmunity in Ghanaian type diabetes patients a casecontrol study BMC Endocr Disord M B T G M P A P PC W Gender differences in thyroid system functionrelevance to bipolar disorder and its treatment Bipolar Disord “ Microvascular Complications and Foot Care Standards of medical Carein Diabetes2018 Diabetes Care 201841S105“s118 Yasuda T Kaneto H Kuroda A Yamamoto T Takahara M Naka T et alSubclinical hypothyroidism is independently associated with albuminuria inpeople with type diabetes Diabetes Res Clin Pract 201194e75“ 0c"
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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 flux 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 flux 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 signaling“cancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdaci™sheatshock 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 iscontextspecificnkg2dls 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 ofspecific 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 find 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 findingssuggest 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 scientific 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 d61345aminoimidazole4carboxamide2deoxydglucosetrifluoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulfide 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 gfpmycmicaˆ— and micaˆ— vectors containingthe coding sequences of micaˆ— or micaˆ— 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 micafirefly luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c o™callaghan university of oxfordoxford united kingdom from tocris biosciencepurification 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 mccoy™s 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoy™s 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 finger 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 confirmed 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 followingmanufacturer™s protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were confirmed by sequencing at eurofins mwgoperons luxembourg colonies were amplified and plasmidswere purified 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 amplified and dna waspurified 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 final volume of µl the dna mixture was complexed with µl 2x hbsunder constant air flow 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 micaˆ— and micaˆ—constructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbriefly dnarna were prepared in µl cacl2 25mand adjusted to a final 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 flow cytometry using accuri c6 flow 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 flow 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 manufacturer™s 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 flow cytometerwas assessed by accurate mass and retention time amrt plusfragment identification 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 confirmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturer™s 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 profilingto 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 filter 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 flight 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 inlinefilter 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 flowrate 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 lchrmsfiles were independently processed with an inhouse developedpcdl library for polar metabolites using profinder version b06agilent technologies identification of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular flux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density — cellswell ˆ¼ 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 flow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modified 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 scientificwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientific 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 manufacturer™s 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 flow 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 fluorescently 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 nkg2d“fc receptor 1299nk rd systemsand igg1“fc 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 fluorescent intensity mfi values aredisplayed in figures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and purified on directzol spincolumns zymoresearch irvine ca united states according to manufacturer™sprotocol 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 firefly 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 firefly 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 transfectionefficiency firefly 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 pcramplified 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 flow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were filtered 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 identified using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantification and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical significance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in figure 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 significance was determined by ˆ—p ˆ—ˆ—p and ˆ—ˆ—ˆ—p ˆ—ˆ—ˆ—ˆ—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 confirm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspecifically to mgat5modified nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modifiednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modification 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 specificity 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 significant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspecific 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 transfectionconfirming 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 deficiency 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 micaˆ— and micaˆ—are distinctly regulated posttranslationally and althoughmicaˆ— 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 offluorescently labeled lpha mgat5 modifications or epha mgat3 modifications on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by flow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean fluorescence 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 signifies 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
properly citedIntroduction Endogenously produced antiganglioside antibodies could aï¬ect the evolution of cutaneous melanoma Epidemiologicaland experimental evidence suggest œchronic ‚ammation to be one of the hallmarks in skin cancers The aim of the study was tocharacterize the relation between antiganglioside antibodies and ‚ammation in cutaneous melanoma focusing on gangliosidesGM1 GM2 GM3 GD1a GD1b GT1b GQ1b Material and Method We performed an observational study that included subjects subdivided into three groups patients with metastatic melanoma cases patients with primary melanoma casesand healthy subjects subjects The assessment of antiganglioside antibodies IgG and IgM classes against GM1 GM2GM3 GD1a GD1b GT1b GQ1b was performed using immunoblot technique EUROLine kit Results The presence of IgGand IgM antiganglioside antibodies in primary melanoma was as follows antiGM1 and GM2 and GM3 and GD1a and GD1b and GT1b and GQ1b and In metastaticmelanoma the level of antiganglioside antibodies was significantly lower compared with primary melanoma p while inthe control group they were absent Antiganglioside antibodies antiGM1 and GD1a were positively correlated while antiGM3GD1b and GT1b were negatively associated with the ‚ammatory markers interleukin IL8 and C reactive protein CRPConclusions Tumour ganglioside antigens generate an immune response in patients with primary melanomas The host™s ability toelaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a dangersignal from the tumour microenvironment Antiganglioside antibodies associated with ‚ammation markers could be used asdiagnostic monitoring and treatment tools in patients with cutaneous melanoma IntroductionGangliosides are a group of bioactive glycolipids located onthe outer face of cell membranes These glycolipids play amajor role in cell proliferation diï¬erentiation migrationapoptosis signal transduction cell adhesion modulatinggrowth factor or hormone receptor antigen recognition protein trafficking viral transformation and oncogenesis [“]Atypical expression of some ganglioside antigens associatedwith certain tumours neuroblastomas melanomas gliomaslymphomas small cell lung cancer and prostate cancer andfurthermore could play an importantrole in cancer 0cJournal of Immunology Researchimmunotherapy [“] Gangliosides that are released inextracellular spaces could have dual action antitumor andprotumour eï¬ect [“] Data regarding the endogenousimmune response directed toward tumour gangliosides andthe significance of this response are limited A series of studiesperformed in in vivo experimental models and in vitro inmurine and human cancer cells have shown that monoclonalantiganglioside antibodies have antitumor potential Theseantibodies exert numerous antitumor eï¬ects through variousmechanisms An important mechanism is the translocationof gangliosides from the plasmatic membrane into theintracellular spaces so binding of antibodies to the surfaceof the tumor cells and complement activation that leads to celllysis mediated by complementdependent cytotoxicity andantibodymediated cellular cytotoxicity [ ] Antiganglioside antibodies modulate ceramide synthesis [ ]reception and transduction of the cytotoxic signal [] theyare involved in suppression or induction of cell death throughdiï¬erent pathways apoptosis necrosis oncogeneslike structural and functional changes of mitochondria accumulationof reactive oxygen species acetylation of gangliosides accumulation of sphingosine sphingamine ceramides [ ]Proteomic studies showed that antiganglioside antibodiescould induce changes like the disruption of signalling systemsP38MAPK PARP JNK123 METc ERK12 P13KAKTand FAK modulation of the level and function of transcription factors P53 SP1 MYCN and HSF1 regulating the balance between apoptosisinducing and apoptosissuppressingfactors cysteineaspartylproteases Bax Bcl2 [ “]These antibodies stimulate the cytotoxicity of chemotherapeutic drugs and small molecule inhibitors [ ] As a result antiganglioside antibodies could be used as diagnostic monitoringand treatment tools in cancer patients [ ]Ganglioside levels are increased in malignant melanocytes and represent an important topic of research [ ]Several researchers have emphasized the role of glycolipidsas markers of melanoma A study analysing the expressionof gangliosides in melanocyte lines and melanoma cell linesfound out an increased expression of GD3 synthase genesin melanoma cells but not in melanocytes The same resultswere obtained for GM2GD2 synthase [] It seems thatgangliosides induce cell proliferation and invasion throughp130Cas and paxillin in melanoma cells []Inflammatory mechanisms play an important role inmelanoma Multiple studies have shown that plasma levelsof C reactive protein CRP increase during tumor proliferation and several relations have been evaluated CRPsurvivalrelationship CRPresponse therapy CRP‚ammationNowadays CRP is considered a true marker for assessing‚ammation in melanoma as well as a marker for responseto treatment Prospective studies have provided consistentresults in the predictive value of CRP in neoplastic diseaseproving high sensitivity and specificity [] In addition inmelanoma elevated levels of CRP may reflect the amountand activity of circulating pro‚ammatory cytokines eginterleukin IL8 IL8 plays a crucial role in regulating cellfunction for host defence and for developing natural immunity [ ] Moreover IL8 is released by various cell typesincluding polymorphonuclear neutrophils PMNs monoSerum dilution Incubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceIncubation with conjugated enzyme degrees min with balanceWashing with universal tampon times min each with balanceIncubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceEUROLine scan evaluationFigure Antiganglioside detectioncytes T lymphocytes and endothelial cells upon exposureto ‚ammatory stimuli Melanoma cells have been reportedto express IL8 and this ‚uences their oncogenic properties[ ] IL8 follows the evolution of melanoma progression and regression under treatment reflecting the stage ofthe disease [“]Based on these accumulating data we have investigatedantiganglioside antibodies in correlation with other ‚ammatory markers IL8 CRP and the clinical evolution of the melanoma patients Clarifying these relations could significantlyimprove the prediction of clinical outcomes Furthermore itcan lead to the development of appropriate therapeutic strategies in patients with cutaneous melanoma Material and Method Patients We performed an observational prospectivestudy during years in Clinical Hospital for Infectious andTropical Diseases œVictor Babes”Dermatology Department Bucharest The study was approved by the Ethics Committee of the Hospital All participants agreed to be includedin research studies without prejudice of the diagnosis orpersonal image and signed the informed consent accordingto the Declaration of HelsinkiThe study included adult patients with cutaneous melanoma with no other pathologies and no treatment for theprimary disease Exclusion criteria were age under yearspregnancy alcohol use melanoma under treatmentWe performed an observational study that included subjects subdivided in three groups patients with metastaticmelanoma cases patients with primary melanoma cases and healthy subjects with matching sex and age subjects Patients were selected and examined accordingto ESMO Clinical Practice Guidelines for melanoma 0cJournal of Immunology ResearchTable Test strips coated with parallel lines of purified antigensAntigenGM1GM2GM3GD1aGD1bGT1bGanglioside typeSourceMonosialoganglioside GM1Monosialoganglioside GM2Monosialoganglioside GM3 Dog erythrocytesBovine brainBovine brainDisialoganglioside GD1aDisialoganglioside GD1bTrisialoganglioside GT1bBovine brainBovine brainBovine brainGQ1bTetrasialoganglioside GQ1bBovine brainStructureGal3GalNAc4[Neu5Ac3]Gal4GlcCerGalNAc4[Neu5Ac3]Gal4GlcCerNeu5Ac3Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac3]Gal4GlcCerGal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac8Neu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCer Glcglucose Gal galactose GalNAc Nacetylgalactosaminediagnosis based on clinical histopathological immunohistochemical and imagistic data All the events related to theprogression of the disease were recorded relapse metastasisneurotoxicity hyper reactivation of the immune system upontreatment The group characteristics were similar for age andsex the primary melanoma group included women and men with a mean age of ± years the metastaticmelanoma group included women and men with a meanage of ± years and the control group included women and men with mean age of ± years Materials and Reagents In this work the assessment ofantiganglioside antibodies was made by the immunoblottechnique using EUROLine kits Figure This methodallows the evaluation of antibodies IgG and IgM classesagainst GM1 GM2 GM3 GD1a GD1b GT1b and GQ1bfrom serumplasma The kit contains strips marked withpurified antigens Table Theevaluation ofantigangliosideantibodies wasperformed using the EUROLine Scan software After readingthe signal intensity on the strips marked with ganglioside antigens the results were evaluated and the results are presented asoptical sensibility The assessment of IL8 was performed bythe ELISA method using Enzo Life Science reagents withTECAN analyser and the results are presented as pgdl CRPwas assessed by immunoturbidimetry using Human reagentsand HumaStar300 analyser the results are presented as mgdl Statistical Analysis All the results were analysed usingIBM SPSS Statistics We evaluated the normality of datadistribution using the KolmogorovSmirnov test The variationbetween groups was determined using the parametric teststtest when two groups were compared or ANOVA test whenmore groups were compared and nonparametric tests likeMannWhitney or Wilcoxon The correlation between groupswas evaluated using linear regression and Pearson coefficientp was considered with statistical significance ResultsAntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b autoantibodies determined in primary metastaticmelanoma had diï¬erent serological profiles compared tothe control group The presence of IgG and IgM antiganglioside antibodies in primary melanoma was as followsantiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and antiGQ1b and Inmetastatic melanoma IgG and IgM antiganglioside antibodies had the following profile antiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and and antiGQ1b and In the control group antiganglioside autoantibodies were absentThe assessment for IgG antiGM1 antiGM2 antiGM3antiGD1a antiGD1b antiGT1a and antiGT1b showedextremely low signal intensity in all groups Figure Whencomparing the mean of signal intensity for IgG no statisticaldiï¬erences were observed between groups We obtained a statistically significant diï¬erence in IgM antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1a and antiGT1b when comparing primary melanoma respectively metastatic melanoma to the control group and once more whencomparing primary versus metastatic melanoma Table To evaluate if the presence of IgM antibodies was associatedwith melanoma development we determined their relation to‚ammatory factors IL8 and CRP recommended by AJCCfor melanoma staging Table IL8 levels were statistically significantly increased in primary melanoma ± pgmland in metastatic melanoma ± pgml when compared with the control group ± pgml CRP levelswere found in primary ± ngml and metastaticmelanoma ± ngml significantly higher when compared with the control group ± ngml IL8 andCRP had no statistically significant variation when comparedto primary versus metastatic melanoma groups Positive correlations with statistical significance were determined betweenantiGM1 and CRP respectively IL8 between antiGD1aand CRP respectively IL8 Figure Negative significant correlations were observed between antiGM3 antiGT1b andCRP respectively IL8 Figure High levels of CRP and IL were associated with an increase in antiGM1 antiGD1aand a decrease in antiGM3 antiGM2 antibodies of IgM typeTable DiscussionsMelanoma the most aggressive skin tumour is a multifactorial cancer being the result of the interplay between geneticimmunological and environmental factors [“] Gangliosides due to their expression on tumor cells have beeninvolved in tumor biology and immunogenicity and hence 0cJournal of Immunology ResearchAntiGM1 IgM and IgG signalintensity in all groups AntiGM2 IgM and IgG signalintensity in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM1 IgM Ž AntiGM1 IgGaAntiGM3 IgM and IgGintensity levels in all groups AntiGM2 IgM Ž AntiGM2 IgGbAntiGD1a IgM and IgGintensity signal in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM3 IgM Ž AntiGM3 IgGcControlPrimary melanomaMetastatic melanomaAntiGD1b IgM and IgG signalintensity in all groupsControlPrimary melanomaMetastatic melanomaAntiGD1b IgM Ž AntiGD1b IgGAntiGD1a IgM Ž AntiGD1a IgGdAntiGT1b IgM and IgGintensity levels in all groups AntiGT1b IgM Ž AntiGT1b IgGefAntiGQ1b IgM and IgGintensity levels in all groupsControlPrimary melanomaMetastatic melanomaAntiGQ1b IgM Ž AntiGQ1b IgGgFigure Antiganglioside signal intensity in all groups ˆ—p have been considered as targets for cancer immunotherapy[“] The probability thatsome tumourassociatedganglioside determinants induce a human immune responsegenerated much interest in medical research [ “]If endogenously synthesized antiganglioside antibodies reactonly with human cancer cells these antibodies could play animportant role in the host™s protective immunity to thetumor There is little information about quantitative variations of serum antigangliosides their origin and progressionof melanoma Tumour ganglioside antigens generate a significantly increased immune response in patients with primarymelanoma versus metastatic melanoma In our study thehost™s ability to generate an early antiganglioside responseis supported by a significantly increased titter of IgMantibodies in patients with primary versus metastatic melanoma and the control groupfor antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1b and antiGQ1b Figure The range of antiganglioside antibodiescould serve as an indicator of diï¬erentiation between patientswith primary melanoma and metastatic melanoma Based on 0cJournal of Immunology ResearchTable IgM antiganglioside signal intensity in all groupsAntibodies Classp significancePM vs MM PM vs control MM vs controlIgMAntiGM1IgMAntiGM2IgMAntiGM3AntiGD1aIgMAntiGD1b IgMAntiGT1bIgMAntiGQ1b IgMMM metastatic melanoma PM primary melanoma psignificancestatisticalTable IL8 and CRP in all studied groupsStudy groupPrimarymelanomaMetastaticmelanomaControlgroupCRPmgdl ± ± ± psignificance”IL8pgml ± ± ± psignificance”our findings we estimate that the levels of the antigangliosideantibodies could provide information regarding the clinicalstaging of melanomaIn addition the capacity of patients to develop an antiganglioside response in the early stage of development of melanoma could be understood as a mean of defence of the bodythrough eliminating a danger signal from the tumour microenvironment represented by the stimulation of glycosphingolipid synthesis [ ] Synthesis of antiganglioside antibodiescould confer a survival advantage in patients with primarymelanoma [] In metastatic melanoma patients we observeda reduction of antiganglioside antibody synthesis a result thatcould suggest the immunosuppressive eï¬ect exerted by theoverproduction of gangliosides associated with tumour metastasis andor due to the overall decreased immune responseIt has been shown in a previous study that in patients withuntreated primary melanoma there is a significant statisticalcorrelation between antiGM1 type IgM level and clinicalstage of the disease Breslow index Clark level tumour localization histologic type presenceabsence of ulceration [ ]In our study patients with cutaneous melanoma had detectable levels of antiGM1 in primary stages The presence of apositive significant relationship between IgM antiGM1 leveland IL8 and CRP in our study justified our statement regarding the involvement of these antibodies in tumour proliferation by stimulating ‚ammation [ “] The presentstudy is the first one that evaluated the relation betweenantiganglioside antibodies and IL8 and CRP based on theirrole in melanoma diagnosis progression and outcome [“] in metabolic disorders [ ]Previous studies in patients with prostate cancer [] orsarcoma [] have shown that antiGM1 antibodies had nodiagnostic or prognostic value in these pathologies In patientswith diï¬erentiated thyroid cancer antiGM1 type IgG andIgM were associated with carcinogenesis but the lack of correlation between antibody level and clinical status indicated thatantiGM1 had no diagnostic value in diï¬erentiated thyroidcancer []Another study performed by our group showed thatpatients with primary melanoma with a high level of IgMantiGM3 had a favourable prognosis compared withpatients displaying a low antibody titer [] In a study onpatients with primary untreated melanoma stages I andII lymph nodes clear of metastasis it was shown that theantiGM2 antibody titer for IgMtype was not diï¬erentiatedin correlation to the tumor thickness For antiGM3 it wasobtained a direct relationship between the serum titer andthe thickness of the tumor [] AntiGM2 and antiGM3antibodies have no diagnostic significance in thyroid cancerdue to the low prevalence of these antibodies [] In ourpresent study antiGM3 negative correlations with IL8and CRP are suitable with the hypothesis that patients withprimary melanoma with a high level of IgM antiGM3 havea favourable prognosisGD1a was thought to generate an immune response inpatients with earlystage melanoma [] In patients withT1T2 stage prostate cancer there were identified increasedIgM antiGD1a values compared to the T3T4 stage whichsustains the development of an early endogenous immuneresponse able to eliminate the danger signal from the tumormicroenvironment These data support the role of antiGD1ain the early diagnosis of localized prostate disease [] TheantiGD1a IgMtype titer was defined as a negative predictivefactor of survival in patients with soft tissue sarcoma [] andin patients with primary melanoma [] In patients™ serumdiagnosed with ovarian cancer an increased titer of IgMantiGD1 was found the authors pointing out that theseantibodies could represent immunological markers associated with ovarian cancer progression [] In cutaneousmelanoma IgM antiGD1a could be considered a markerassociated with melanoma progression based on the negativecorrelation with CRP and IL8 as shown in our studyThe antiGD1b immune response in patients with gastricneoplasm can be used as a prognostic marker [] On thecontrary the lack of correlation between the presence ofantiGD1b and the clinical status of patients with thyroidcancer has indicated that antigangliosides do not have diagnostic significance in this neoplasm []The antiGT1b titer can be an overall positive factor associated to global survival in sarcoma [] AntiGD1b GT1band GQ1b antibodies that are negatively correlated with IL8and CRP suggest that they could indirectly suppress tumorgrowth and angiogenesis AntiGD1b GT1b and GQ1bIgM type ‚uence the progression of melanoma [ ]soft tissue sarcomas [ ] Ehrlich subcutaneous solidtumors [ ] Ehrlich carcinomas accompanied by ascites[ ] and gastric cancer []Our study limitations could be considered the semiquantitative assessment method of antiganglioside antibodies as aquantitative determination technique could oï¬er more sensitive data ing the door for further studies One more 0cJournal of Immunology ResearchMGitnAMGitnAaDGitnA CRP CRP CRP MGitnAMGitnAaDGitnAIL8IL8IL8Figure AntiGM1 antiGM3 and antiGD1a in relation to CRP and IL8 in the primary melanoma groupimportant limitation of the study is that we evaluated thecorrelation between antiganglioside antibodies and ‚ammation markers in melanoma IL8 and CRP only after orbetween the surgical treatment of melanoma newer therapiesbeing also in place This could be the first study which otherresearchers and clinicians can use and analyze in order toevaluate the ‚uence of diï¬erent melanoma treatments onantiganglioside antibodies The pathogenic mechanismsinvolved in melanoma are complex [“] therefore theevaluation during the followup period of melanoma patientsat diï¬erent points is needed for a better antigangliosideprofile characterization 0cJournal of Immunology ResearchTable AntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b relation with ‚ammatory markers in primary melanomagroupAntiGM1AntiGM2AntiGM3AntiGD1aAntiGD1bAntiGT1bAntiGQ1bCRPr p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p IL8r p ‰¤ r ˆ’p r ˆ’p r p ‰¤ r ˆ’p ‰¤ r ˆ’p ‰¤ r ˆ’p ConclusionsAntiganglioside antibodies antiGM1 and GD1a werepositively correlated while antiGM3 GD1b and GT1bwere negatively associated with the ‚ammatory markersIL8 and CRP The host™s ability to elaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a danger signal from thetumour microenvironment Moreover our results suggest thattumour ganglioside antigens generate a significantly increasedimmune response in patients with primary versus metastaticmelanoma Antiganglioside antibodies associated with ‚ammation markers could be used as diagnostic monitoring andtreatment tools in patients with cutaneous melanomaData AvailabilityThe data used to support the findings of this study areincluded within the articleConflicts of InterestThe authors declare no conflicts of interestAuthors™ ContributionsAll authors have equally contributed to the writing and editing of the manuscriptAcknowledgmentsThis research and article processing charges were funded by agrant of the Romanian Ministry of Research and InnovationCCCDIUEFISCDI project number 61PCCDI2018 PNIIIP112PCCDI2017034References[] YH Xu S Barnes Y Sun and G A Grabowski œMultisystem disorders of glycosphingolipid and ganglioside metabolism Journal of Lipid Research vol no pp “ [] I Horwacik and H Rokita œTargeting of tumorassociatedgangliosides with antibodies aï¬ects signaling pathways andleads to cell death including apoptosis Apoptosis vol no pp “ [] J L Daniotti R D Lardone and A A Vilcaes œDysregulatedexpression of glycolipids in Tumor cells from negative modulator of Antitumor immunity to promising targets for developing therapeutic agents Frontiers in Oncology vol p [] I Nicolae A Caragheorgheopol S Schipor œGnagliosides and sex hormones in human melanoma Acta Endocrinologica vol no pp “ [] Y Ohmi M Kambe Y Ohkawa œDiï¬erential roles ofgangliosides in malignant properties of melanomas PLoSONE vol no article e0206881 [] M H Ravindranath S Muthugounder N Presser A D Santin R S Selvan and D L Morton œGanglioside GD1a present in ovarian cancer cells ascites and sera of patients elicitsendogenous IgM response Proceedings of the American Association for Cancer Research vol p [] V B Doronin T A Parkhomenko M Castellazzi et alœComparison of antibodies hydrolyzing myelin basic proteinfrom the cerebrospinal fluid and serum of patients with multiple sclerosis PLoS One vol no article e107807 [] C D Ene and I Nicolae œGangliosides and Antigangliosidesin Malignant Melanoma Melanoma Current Clinical Management and Future Therapeutics M Murph Ed MandiMurph Intech ISBN [] N AlvarezRueda S Leprieur B Clemenceau œBindingactivities and antitumor properties of a new mousehumanchimeric antibody specific for GD2 ganglioside antigen Clinical Cancer Research vol no pp 5613s“5620s [] K 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production by isolated human hepatocytes American Journal ofPhysiologyEndocrinology and Metabolism vol no pp E720“E726 [] M Bickel œThe role of interleukin8 in ‚ammation andJournal of Periodontologyregulationmechanisms ofvol no pp “ [] M Neagu C Constantin and S Zurac œImmune Parametersin The Prognosis and Therapy Monitoring of Cutaneous Melanoma Patients Experience Role and Limitations BioMedResearch International vol Article ID pages[] S R Georgescu M R Ioghen M I Sarbu œBiologicaltherapy in the treatment of melanoma Journal of Mind andMedical Sciences vol no pp “ [] A V Dumitru M Tampa S R Georgescu œImmunohistochemical mismatch in a case of rhabdomyoblastic metastaticmelanoma Romanian Journal of Morphology and Embryology vol no pp “ [] S N Pavri J Clune S Ariyan and D Narayan œMalignantMelanoma Plastic and Reconstructive Surgery vol no pp 330e“340e [] M Rastrelli S Tropea C R Rossi and M Alaibac œMelanoma epidemiology risk factors pathogenesis Diagnosis andClassification In Vivo vol no pp “ [] L LugovićMihić D Ćesić P Vuković G Novak BilićM Å itum and S Å poljar œMelanoma development currentknowledge on melanoma pathogenesis Acta Dermatovenerologica Croatica vol no pp “ [] M Costache A V Dumitru O M Pătraşcu œA challenging case of ocular melanoma Romanian Journal of Morphology and Embryology vol Suppl pp “ [] R Ancuceanu and M Neagu œImmune based therapy for melanoma Indian Journal of Medical Research vol no pp “ [] C A Perez M H Ravindranath D Soh A Gonzales W Yeand D L Morton œSerum antiganglioside IgM antibodies insoft tissue sarcoma clinical prognostic implications CancerJournal vol no pp “ [] B Mondal and S Sahal œInhibition of subcutaneous growth ofEhrlich ascites carcinoma EAC tumor by postimmunizationwith EACcell gangliosides and its antiidiotype antibody inrelation to tumor angiogenesis apoptosis cell cycle and ltration of CD4 CD8 lymphocytes NK cells suppressorcells and APCcells in tumor Indian Journal of ExperimentalBiology vol no pp “ [] S Sahal and S Mondal œSupression of Ehrlich subcutaneoussolid tumor growth by immunization with ganglioside GT1bof its origin its IgM antibody or antiidiotype antibody Journal of Experimental Clinical Cancer Research vol no p [] M M Konstandoulakis K N Syrigos M LeandrosA Charalabopoulos A Manouras and B C GolematisœAutoantibodies in the serum of patients with gastric cancertheir prognostic importance Hybridoma vol no pp “ [] M H Ravindranath S Muthugounder X Ye and D L Morton œInnate immune response to gangliosides of primary melanoma favors danger hypothesis Proceedings of the AmericanAssociation for Cancer Research vol p [] M H Ravindranath S Muthugounder and N Presser œGanglioside signatures of primary and nodal metastatic melanomacell lines from the same patient Melanoma Research vol no pp “ [] A Lewartowska T Pacuszka G Adler M Panasiewicz andW Wojciechowska œGanglioside reactive antibodies of IgGand IgM class in sera of patients with diï¬erentiated thyroidcancer Immunology 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Nicolae œ25OHvitamin D and interleukin8 emerging biomarkers in cutaneous melanoma development and progression Mediators ofInflammation vol Article ID pages [] N R Sproston and J J Ashworth œRole of Creactive proteinat sites of ‚ammation and infection Frontiers In Immunology vol p [] A E Anghel C D Ene M Neagu and I Nicolae œThe relationship between interleukin8 and Ki67 in cutaneous malignant melanoma HVM Bioflux vol no pp “[] C D E Nicolae and I Nicolae œInterleukin 8serumconcentration but not lactate dehydrogenase activity positively correlates to CD34 antigen in melanoma tumors Journal of 0cJournal of Immunology ResearchImmunoassay and Immunochemistry vol no pp “ [] A E Anghel C D Ene I Nicolae V A Budu C Constantinand M Neagu œInterleukin major player in cutaneous melanoma metastasic process Romanian Biotechnological Letters vol no pp “ [] M Neagu œMetabolic traits in cutaneous melanoma Frontiers in oncology vol p [] C D Ene A E Anghel M Neagu and I Nicolae œInterleukin and 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"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjonThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partner™s occupation and economic statusPatient™s occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatient™s marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patient™s hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patient™s family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants™ hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ‰¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ‰¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet 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 Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc„¢ wwwwjon 0c"
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" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearman™s correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis 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 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturer™s instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time × power time × power–¼–¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci “ pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci “ pgml and pgml ci“ pgml respectively the median level of il p40 before the mwa treatment was pgml ci “ pgml there was a slight increase to pgml ci “ pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci “ pgml and increasedto pgml ci “ pgml days afterthe mwa treatment and to pgml ci “ pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci “ pgml there was a significant increase at h postmwa with a median level of pgml ci “ pgml the median level ofil1 before the mwa treatment was pgml ci “ pgml and a significantincrease wasnoted days after the mwa treatment pgml ci “ pgml the median level of il6before the mwa treatment was pgml ci“ pgml and significantly increased daysafter the mwa treatment pgml ci “ pgml the median level ofil8 before themwa treatment was pgml ci “ pgml and increased significantly to pgml ci“ pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci “ pgml and increasedsignificantly days after the mwa treatment pgml ci “ pgml the median level oftnfα before the mwa treatment was pgml ci “ pgml and increased significantlyto pgml ci “ pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ h postmwa pgml ci “ ci “ –¼ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa –¼ 2fold vs premwa days postmwa pgml ci “ ci “ ci “ ci “ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ days postmwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 it™s mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaˆ’energyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearman™s rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wasœcancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as œdifferentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmed™s work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors™ contributionsjz conceptualization data curation writing“original draft and writing“review and editing ql conceptualization and writing“review and editingmm conceptualization and writing“review and editing brconceptualization and writing“review and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writing“review and editing rl conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the œsix one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett “bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144“rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol “yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res “lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218“jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine “ gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol “ ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology “ chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc “ huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres “ alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res “kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a “ onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother “ yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget “ yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490“erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol “ den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res “ zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology “ zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim
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EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garc­a1 Jose I Colomer1 Juan A Jover34 Benjam­n Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was “ days The median length of stay was “ days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject –º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add –º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis –º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments –º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Cl­nico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the χ2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of “ days The median length of stay was “ days table At admission the median haemoglobin was “ gdL and the median total lymphocyte count was “ ngmL The median D dimer value was “ ngmL In of patients median interleukin IL6 levels were “ pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patient™s condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of “ years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogren™s syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI “ p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI “ p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI “ p09 and NSAIDs OR CI “ p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI “ p016 and non anti TNF vs none OR CI “ p03DISCUSSIONOur study aims to shed light on rheumatologists™ concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9“ and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRD“COVID19 patientsAIRD“COVID non admitted patientsAIRD“COVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogren™s syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexate“leflunomide“azathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count —109L Lymphocyte count —109L CRP mgdL LDH UL Platelet count —109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes “ Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ‰¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 “ “ “ “ “ “ “ “ “ ““““““““““““ Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug““““““““““““ ““ Methotrexate“leflunomide“azathioprine Sulfasalazine Antimalarial agents““““““““““ None Anti TNF agents Other biologics““As for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patient™s history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP value“““““Gender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjam­n Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Cl­nico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin “ COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis “ Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art
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Understanding of the RelevantRole of LINE1 Retrotransposition inHuman Disease and ImmuneModulationXiao Zhang12 Rui Zhang12 and Jinpu Yu12 Cancer Molecular Diagnostics Core Tianjin Medical University Cancer Institute Hospital National Clinical ResearchCenter of Caner Key Laboratory of Cancer Prevention and Therapy Key Laboratory of Cancer Immunology and BiotherapyTianjin China Tianjin™s Clinical Research Center for Cancer Tianjin ChinaLong interspersed nuclear element1 LINE1 retrotransposition is a major hallmark ofcancer accompanied by global chromosomal instability genomic instability and geneticheterogeneity and has become one indicator for the occurrence development andpoor prognosis of many diseases LINE1 also modulates the immune system andaffects the immune microenvironment in a variety of ways Aberrant expression of LINE retrotransposon can provide strong stimuli for an innate immune response activatethe immune system and induce autoimmunity and ‚ammation Therefore inhibitionthe activity of LINE1 has become a potential treatment strategy for various diseasesIn this review we discussed the components and regulatory mechanisms involved withLINE1 its correlations with disease and immunity and multiple inhibitors of LINE1providing a new understanding of LINE1Keywords retrotransposons LINE1 regulatory mechanisms cancer immune inhibitorINTRODUCTIONLong interspersed nuclear elements LINEs are the only autonomous and active retrotransposonswhich include LINE1 LINE2 and LINE3 Cordaux and Batzer de Koning Also “ of LINE2 and LINE3 sequences in the human genome are as a truncated molecularfossil Doxiadis Ardeljan LINE1 retrotransposons are one of the mostabundant and eï¬ective classes of mobile DNAs that account for of the human genomeLander Hancks and Kazazian Fulllength LINE1 is “ kb and containsa 5cid48untranslated region 5cid48UTR Swergold two reading frames ORF1 and ORF2and a 3cid48UTR punctuated with a polyA tract Babushok and Kazazian Beck Denli revealed a new reading frame ORF0 It is located in the 5cid48UTR of theLINE1 transcript and on the strand opposite of the ORF1 and ORF2 structural genes Antisensepromotor ASP can initiate fusion transcripts and regulate ORF0 to enhance LINE1 mobilityRomanGomez Weber Criscione Both ORFs are required for LINE1 retrotransposition process ORF1 encodes an RNAbindingprotein named ORF1P that has nucleic acid chaperone activity and ORF2 encodes a proteinnamed ORF2P that has endonuclease and reversetranscriptase activities Mathias Feng The first step occurs when RNA polymerase II binds to the 5cid48UTR promoterregion of LINE1 and mediates the transcription of fulllength mRNA of LINE1 Lavie The LINE1 mRNA is exported to the cytoplasm where ORF1 and ORF2 are translatedand combined to form a ribonucleoprotein RNP p The RNP is then incorporated intoEdited byTrygve TollefsbolThe University of Alabamaat Birmingham United StatesReviewed bySandra Rose RichardsonThe University of QueenslandAustraliaApiwat MutiranguraChulalongkorn University ThailandJohn LaCavaThe Rockefeller UniversityUnited StatesCorrespondenceJinpu YuyujinputjmuchcomSpecialty sectionThis was submitted toEpigenomics and Epigeneticsa section of the journalFrontiers in Cell and DevelopmentalBiologyReceived April Accepted July Published August CitationZhang X Zhang R and Yu J New Understanding of theRelevant Role of LINE1Retrotransposition in Human Diseaseand Immune ModulationFront Cell Dev Biol 103389fcell202000657Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasethe nucleus and the ORF2P endonuclease in the RNP identifiesand cuts specific sequences on the bottom DNA strand atthe consensus site 3cid48ˆ’AATTTTˆ’5cid48 Subsequently the free 3cid48hydroxyl generated at the fracture is utilized by the ORF2P andLINE1 mRNA in the RNP is used as the template for reversetranscription to produce the complementary DNA of the LINE gene Wei Hancks and Kazazian Wang andJordan The distribution of LINE1 in the human genomeis selective LINE1 endonuclease activity and DNA replicationdetermine LINE1 insertion preference Flasch Forexample LINE1 preferentially inserts into nucleosomedepletedDNA primarily as a result of its ATrich sequences Sultana The direction of the DNA replication fork aï¬ects LINE insertion preference because the cleaved strand is usually thelagging strand templateLINE1 elements play a crucial role in the course of speciesformation and evolution On one hand derepressed LINE1functions as a driver of many diseases and even a diagnosticmarker for some diseases Pedersen and Zisoulis On theother it can aï¬ect the developmental processes and ‚uencethe behavior by generating multiple gene products and causingvariable deleterious eï¬ects on the structure of the host genomethrough new insertions deletions and recombinations GarciaPerez LINE1 RNA and protein overexpression isrelated to apoptosis DNA damage and repair cellular plasticityand stress responses and can even promote tumor progressionMorrish Belgnaoui SinibaldiVallebona DNA damage caused by genomewide or intersperse repetitive sequences hypomethylation caninduce ‚ammatory microenvironment Lindqvist Teerawattanapong Here we reviewed the correlationbetween LINE1 and disease as well as immune systemmeanwhile conducted a new exploration in LINE1 inhibitors bycombining its regulation mechanismsFigure shows the relative positions of the 5cid48 untranslatedregion 5cid48UTR the reading frames ORF0 ORF1 andORF2 the 3cid48 untranslated region 3cid48UTR and the Poly A tailORF2 encodes endonuclease EN reverse transcriptase RTand cysteinerich domain C Fulllength LINE1 mRNA wasgenerated using the sense promoter at 5cid48UTR The LINE1mRNA is exported to the cytoplasm where ORF1 and ORF2are translated and combined to form a ribonucleoprotein RNPp The RNP is then incorporated into the nucleus andthe ORF2P endonuclease in the RNP identifies and cuts specificsequences on the bottom DNA strand at the consensus site3cid48ˆ’AATTTTˆ’5cid48 Subsequently the free 3cid48 hydroxyl generatedat the fracture is utilized by the ORF2P and LINE1 mRNA in theRNP is used as the template for reverse transcription to producethe complementary DNA of the LINE1 gene Richardson Kazazian and Moran LINE1 AND DISEASELINE1 and CancerWhen LINE1 retrotransposition is out of control it can lead todiseases More than s focusing on LINE1 and cancerare available in the PubMed archive Rodic theglobal hypomethylation ofLINE1 Hypomethylation and Cancergenome promotesThechromosomalinstability genomic instability and geneticheterogeneity because specific changes in DNA methylation canaï¬ect a variety of genome sequences especially the intergenicand intronic regions of the DNA resulting in chromosomeinstability and mutations Wilson LINE1 promoterhypomethylation is a biomarkerfor genomewide DNAhypomethylation which is itself a major hallmark of cancerThayer first demonstrated the methylation statusof LINE1 in cancer cells Since then LINE1 hypomethylationof tumors has attracted widespread attention Thayer LINE1 hypomethylation was reported to be associatedwith poor survival in more than cases of gastric cancersuggesting its potential as a prognostic biomarker Shigaki This phenomenon was also subsequently foundin lung cancer liver cancer esophageal cancer prostate cancerand endometrial cancer Iwagami Kawano Lavasanifar Ogino analyzed colon cancer samples from two independent prospectivecohorts demonstrating a linear correlation between LINE hypomethylation and aggressive tumor behaviorIt hasbeen reported that global DNA hypomethylation promotesaggressive tumor behavior by amplifying oncogenes or throughabnormal expression of microRNAs Baba In esophageal cancer with high mortality and poor endoscopicscreening sensitivity LINE1 hypomethylation can serve as agood diagnostic biomarker thereby improving 5year survivalShah LINE1 hypomethylation can also be seenin some precancerous lesions For examplein colorectalcancer LINE1 hypomethylation had no significant diï¬erencebetween adenomas and cancerous tissues but it was significantlylower in adenomas than in normal tissues Dawwas Therefore LINE1 hypomethylation also can be used as an earlybiomarker for cancerHoweverthere was no significant diï¬erencein thehypomethylation of LINE1 between the blood samplesof patients with leukemia and those of normalsubjectsBarchitta LINE1 Integrations and CancerMany tumor tissues have been found to present a high levelof LINE1 activity that can rapidly increase their copy numberthrough the œcopyandpaste mechanism Dunaeva LINE1 can be used as cisregulatory elements to regulatethe expression of host genes Wanichnopparat Pancancer Analysis of Whole Genomes analysis of cancer genomes from histological subtypes revealed thataberrant LINE1 integrations could lead to gene rearrangementRodriguezMartin LINE1mediated rearrangementcan trigger oncogene amplification In breast cancer Morse andcolleagues first proposed that hypomethylation activates LINE which can utilize the target primed reverse transcriptionpathway to insert into the oncogene MYC causing tumorspecificrearrangement and amplification Morse LINE was found to induce the amplification of CCND1 oncogenein esophageal tumor by inducing the breakage“fusion“bridgeFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human DiseaseFIGURE Structure of LINE1 and LINE1 retrotransposition cyclecycles RodriguezMartin LINE1 can mediatethe deletion of tumor suppressor genes It may be through Xinactivation mechanism that LINE1 mRNA forms facultativeheterochromatin in the inactivated region or LINE1 mRNAand premRNA form RISC complex to degrade complementarymRNA Allen Aporntewan In coloncancer Miki reported that LINE1 insertion disrupts thetumor suppressor gene APC which can lead to gene inactivationMiki In lung squamous cell carcinoma wefound that LINE1 insertion into tumor suppressor gene FGGYpromotes cell proliferation and invasion in vitro and facilitatestumorigenesis in vivo Zhang High Expression of ORF1 and ORF2 ofLINE1 and CancerThe activation of LINE1 increases the translation of ORF1 andORF2 which are not expressed in normal somatic tissues ORF1encodes an RNAbinding protein and high expression level ofORF1 was proved to be more common in most of the cancersand therefore as a diagnostic marker In breast cancer highexpression of nuclear ORF1 is associated with distant metastasisand poor prognosis Harris In highgrade ovariancarcinoma the ORF1 level was high and correlated to the lossof TP53 Rodic The expression of both the LINE1ORF1 and cMet protein was significantly increased and peakedin early stage in ovarian cancer suggesting that LINE1 ORF1significantly activates cMet Ko In tumor cellexperiments increased mRNA and protein expression of LINE1ORF1 can result in significant enhancement in cell proliferationand colony formation Tang It is worth noting thatthe expression of ORF1 was heterogeneous and had histologicalspecificity Cancers originating in the endometrium such asbiliary tract esophagus bladder head and neck lung and colonexhibit ORF1 overexpression whereas other cancers such asrenal liver and cervical cancer show little expression of ORF1Ardeljan Recent studies have shown that an ELISAmethod to measure ORF1 in serum can be better in prostatecancer detection Hosseinnejad ORF2 encodes a protein with reverse transcriptase andendonuclease activities High expression of endonuclease inducesdoublestrand DNA breakage that can aggravate DNA damagerepair and increase genomic instability Kines Reverse transcriptase activation can promote cell proliferationthe noncoding RNAand diï¬erentiation and also altertranscription spectrum and otherepigenetic phenotypesresulting in alterations in cell regulatory networkstumordevelopment and other important pathological processes Rodicand Burns Burns Christian ORF2 canexpress early in the tumorigenesis process as it can be detectedby a highly specific monoclonal antibody mAb chA1L1 in bothtransitional colon mucosa and prostate intraepithelial neoplasiasDe Luca However studies have shown that chA1L1 recognizes both ORF2p and the transcriptional regulatorBCLAF1 so it is not specific Briggs But recentlytumor proteome profiling studies based on mass spectrometryhave shown that ORF2p was difficult to be detected and afteraffinity capture of ORF1p ORF2p has not been detected in stemcell LINE1 proteome analysis Vuong Ardeljan Therefore the detection and application of ORF2 intumors are still worth exploringLINE1 and Metabolic DisordersNew research has shown that LINE1 is also associated with bloodsugar and lipid levels Turcot LINE1 methylation isassociated with type diabetes mellitus T2DM Studies showedthat compared with hypermethylation LINE1 hypomethylationwas associated with a higher risk of worsening metabolic statusindependent of other classic risk factors MartinNunez This discovery highlights the potential role for LINE1DNA methylation as a predictor of the risk of T2DM orFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseaseother related metabolic disorders LINE1 DNA methylation isassociated with increased LDL cholesterol and decreased HDLcholesterol levels and these metabolic changes increase the riskof cardiovascular disease Pearce LINE1 DNAmethylation is also associated with many bloodbased metabolicbiomarkers In fetal neural tissue with neural tube defects it wasfound that the low methylation level of LINE1 was associatedwith the significant reduction of vitamin B12 in maternal plasmaas well as lower folate levels and increased concentrations ofhomocysteine Wang Folic acid and other B vitaminsplay an important role in the biosynthesis of new purines andpyrimidines Therefore the methylation status of LINE1 can be apredictor of some metabolic diseases Current studies have shownthat LINE1 can also regulate metabolism by inserting metabolicgenes It was reported that LINE1 insertions in the FGGY genecan upregulate cytochrome P450 arachidonic acid metabolismand glycerolipid metabolism These metabolic disorders can leadto the occurrence of a variety of diseases and poor prognosisZhang LINE1 and Neurological DisordersLINE1 can aï¬ect the developing brain at diï¬erent stages ofhealth and disease Suarez Ataxia telangiectasiaAT is a progressive neurodegenerative disease caused byataxia telangiectasia mutated ATM gene mutation In researchers found that in nasopharyngeal carcinomas with ATMdeficiency LINE1 retrotransposition increased and ORF2 copynumber increased in AT neurons thus verifying the correlationbetween LINE1 retrotransposition and ATM deficiency Coufal High expression of LINE1 was found in Rettsyndrome caused by mutation of methyl CpG binding protein MeCP2 in the Xlinked gene which was caused by theinclusion of LINE1 5cid48UTR sequence in the MeCP2 targetleading to methylationdependent repression Muotri LINE1 is involved in the aging process In patientswith frontotemporal lobe degeneration LINE1 transcripts werefound to be elevated Li LINE1 hypomethylationhas been observed in most psychiatric studies Increased copynumbers of LINE1 as a result of LINE1 hypomethylation werealso found in patients with schizophrenia bipolar disorder andmajor depressive disorder Liu Li Thelink between LINE1 methylation levels and Alzheimer™s diseaseis still being studiedLINE1 and Genetic DisordersLINE1 is reported to be related to chromosome disordersThe first observation of LINE1 insertion was in whenKazazian observed a new exon of F8 LINE1 insertion inthe Xlinked gene which is a gene encoding coagulation factorVIII in a patient with hemophilia A Kazazian Then a LINE1 insertion was found in the CHM gene of apatient diagnosed with choroideremia The reverse integrationof a LINE1 element into exon resulted in aberrant splicingof the CHM mRNA van den Hurk FurthermoreLINE1 can also promote mobilization of other RNAs intrans Alu and SVA which can be transmobilized leading togene insertions Kemp and Longworth Retrotransposoninsertions were found to account for up to of all NF1mutations Wimmer Neurofibromatosis type I isan autosomal dominant disorder caused by NF1 gene mutationsMessiaen Alu insertion is located bp upstreamof NF1 exon causing the exon to skip and change the reading frame Payer and Burns Only two cases werethought to be a result of independent SVA insertion in SUZ12Paccompanied by 867kb and 1Mb deletions that encompassedthe NF1 gene Vogt In autosomal recessive geneticdisease such as Fanconi anemia caused by SLX4FANCP deficiencyand Aicardi“Goutieres syndrome AGS of threeprime repairexonuclease mutations LINE1 expression was upregulated andpro‚ammatory cytokines were produced through the cGAS“STING pathway Brégnard Suarez LINE1 AND IMMUNE REGULATION OFDISEASELINE1 and Autoimmune DiseaseHypomethylated and highly expressed LINE1 has been foundin autoimmune diseases such as systemic lupus erythematosusSLE Sjögren™s syndrome SS and psoriasis Schulz Yooyongsatit Mavragani LINE1 RNAis characterized by viral RNA and exists as RNP ps whichcan be recognized by RNA sensors and activate innate immuneresponses Mavragani Cell studies demonstrated thatLINE1 activates the production of IFNβ by RNA pathway Zhao When LINE1 retrotransposition was inhibited byRT inhibitors significant reductions were observed in IFNαIFNβ and IFNγ mRNA levels Brégnard LINE1transcripts and p40 protein a ˆ’kDa RNA binding proteinthat LINE1 encodes have been detected in SLE and SS patientsIt has been demonstrated that LINE1 can induce the productionof IFNI in vitro by TLRdependent and TLRindependentpathways Mavragani In MRL autoimmunelymphoproliferative syndrome LINE1 ORF2 encoding an RTand its products are associated with an MHC class I molecule onthe cell membrane Benihoud In Fanconi anemia andAGS LINE1 was found to be associated with the activation of theautoimmune system LINE1 also regulates immunity by actingas a cisregulatory element through the mechanism of LINE1mRNA and premRNA forming RISC complex to degrade thecomplementary mRNA Wanichnopparat LINE1 and Tumor ImmunityIn TCGA cancer samples the scientists measured thetranscriptional activity of pathways and found that of immune pathways were significantly negatively correlatedwith LINE1 Jung LINE1 is inversely correlatedwith the expression of immunologic response genes Less LINE activity was found in tumors with high immune activityIn esophageal cancer tissues scientists found that the LINE1methylation level in tumors was significantly positively associatedwith the peritumoral lymphocytic reaction Kosumi The activities of regulatory T cells and PD1 signaling as reportedin cancer immune evasion and chronic ‚ammatory conditionsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasealso have negative correlations with LINE1 It is reported that thenegative correlation between LINE1 and immune activity maybe caused by the destruction of LINE1 inhibition but the specificmechanism is still unclear LINE1 may also mediate immunetolerance which may change from immune stimulation mode toimmunosuppression mode through continuous IFN signaling ordirectly aï¬ect lymphocyte signalingtumorLINE1 and MetabolismInducedImmunityLINE1 is also associated with blood sugar and lipid levelsAbnormal glucose and lipid metabolism can lead to metabolicreprogramming in tumor cells The most classic metabolismofis Warburg eï¬ect where a large amount ofglucose is absorbed to fulfillthe need for proliferationand produce lactic acid Lunt and Vander Heiden The acidic microenvironment caused by lactic acid leads toimpaired Tcell activation and proliferation prevents NK cellactivation stabilizes HIF1α to stimulate the polarization of anti‚ammatory M2 macrophages and inhibits the production ofIFNγ in tumorltrating T cells Husain Colegio Brand Abnormal lipid metabolism intumor cells also can lead to local immunosuppression in themicroenvironment Hao LINE1 can aï¬ect localimmune homeostasis by inserting elements into metabolismrelated genes FGGY is known to encode a protein thatphosphorylates carbohydrates and is associated with obesity andsporadic amyotrophic lateral sclerosis Zhang LINE retrotransposons suppress FGGY leading to lipid metabolismdisturbance and dietinduced obesity in mice Taylor Lung squamous cell carcinoma patients with L1FGGYtissue have a poor prognosis have low levels of CD3 Tcells and have high levels of CD68 macrophages and CD33myeloidderived cells Zhang L1FGGY alsoregulates the abnormal transcription of cytokines related to theimmunosuppressive micromilieuLINE1 INHIBITIONThe correlation between LINE1 and disease as well as immunitywas analyzed Figure The life cycle of LINE1 providesa plethora of ways to target and inhibit LINE1 expressionBanuelosSanchez The inhibition of LINE1 hasbecome a treatment strategy for some diseasesTargeting LINE1 Methylationa CpGFulllength LINE1 transcription is driven bydinucleotiderich internal promoter Hypomethylation of LINE causes the activation of LINE1 which causes retroelementtransposition and chromosomal alteration Saito The hypomethylation of LINE1 has become an important factorin the occurrence and development of diseases so maintainingthe state of LINE1 methylation has become a key methodfor the treatment of diseases Soy isoflavone supplementationcan regulate the level of LINE1 methylation in head and necksquamous cell carcinoma HNSCC In a clinical trial of patients with HNSCC who took a soy isoflavone supplement mgday orally for weeks before surgery a positivecorrelation was found between LINE1 methylation level anddaily isoflavone intake Rozek Some cellbasedstudies and clinical data have shown that LINE1 dysregulationis associated with tumor drug resistance Zhu Lavasanifar It was found in breast cancer cellstreated with paclitaxel that DNMT3a a member of the DNAmethyltransferase family could enhance the methylation level inthe gene by binding to the inner region of the LINE1 gene andthen upregulate the expression level of LINE1 Downregulatingthe expression of DNMT3a can eï¬ectively inhibit the expressionof LINE1 Wang LINE1 retrotransposon silencedalso through histone modifications Histone demethylaseKDM4B may enhance the LINE1 retrotransposition efficacywhereas depletion of KDM4B reduced itin breast cancerXiang Elevated LINE1 expression was found inPC9 drugtolerant persister DTP cancer cells treated withthe EGFR inhibitor erlotinib HDAC inhibitors can suppressLINE1 in DTP cancer cells Guler Currently DNAmethyltransferase inhibitors and histone deacetylase inhibitorshave entered clinical trials Gaillard Targeting RT ActivityLINE1 elements harbor ORF1 and ORF2 which has reversetranscriptase RT activity and RT inhibition may be anovel noncytotoxic anticancer therapy strategy Sciamanna RT is a key player in retrotransposition andfunctions by transcribing LINE1 mRNA or other RNAs toFIGURE The relationship between LINE1 and diseases and their regulatory mechanismsFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human DiseasecDNA at the integration sites Khalid Specificreverse transcription inhibitorsincluding nevirapine NVRand efavirenz EFV which target the HIV1encoded RT andare currently used in AIDS therapy reduce cell proliferationand promotes diï¬erentiation of a variety of cancer cell linesof unrelated histological origin Mangiacasale Landriscina Sciamanna In vivoassays using murine models inoculated with various humancancer cell lines revealed that daily treatment of animals withEFV significantly delayed the progression of tumors Oricchio NVR and EFV dramatically countered L1FGGYabundanceinhibited tumor growth attenuated metabolismdysfunction and improved the local immune evasion in lungsquamous cell carcinomas Zhang EFV hasrecently undergone a phase II clinical trial in patients withmetastatic prostate cancer Houédé Another RTinhibitor F2DABOs has shown antiproliferative activity innude mice helping to promote cell diï¬erentiation and inhibittumor growth Sbardella Later the nucleosidereverse transcription inhibitor abacavir was also shown to inhibitcell growth migration and invasion Carlini Capsaicin is the main chemical component of Asiasari radixand Capsicum annuum as well as the major component of aChinese traditional herbal medicine Shoseiryuto Friedman Capsaicin suppresses LINE1 by inhibiting theRT activity of LINE1 ORF2P which is the LINE1encodedRT responsible for LINE1 activity Nishikawa A recent study revealed that azidothymidine AZT inhibitsthe RT activity of ORF2P in a fetal oocyte attrition modelExperiments showed that AZTtreated oocytes have a reductionof LINE1 ORF1 ssDNA compared with untreated oocytesTharp It is important to note that RT inhibitorsdo not eliminate the tumor but only control its progressionTherefore in addition to the antiAIDS drugs approved by theFDA the combination of Chinese and western medicine can beregarded as an emerging treatmentCombined ImmunotherapyRecent studies suggest that LINE1 hypomethylation may be apositive indicator of immunotherapy DNA methyltransferaseDNMT is an important epigenetic molecule that catalyzedDNA methylation and can induce the development of varioustumors Downregulating the expression of DNMT can eï¬ectivelyinhibitthe expression of LINE1 Wang SoDNA methyltransferase inhibitors DNMTis play an importantrole in the antitumor process DNMTI can improve tumorimmunogenicity promote NK cells and CD8 T cells toplay a cellmediated cytotoxic role and promote immuneresponse to participate in antigen commission by regulatingimmunosuppressive cells Chiappinelli DNMTican enhance the expression of cancertestis CT antigenmaking the tumor more susceptible to CT antigen vaccine Thecombination of decitabine a DNA methyltransferase inhibitorand cancertestiscancergermline antigen NYESO1 vaccinehas a good therapeutic eï¬ect in the primary treatment ofhuman recurrent epithelial ovarian cancer Odunsi A clinical trial has shown that combination therapywith carboplatin and antiprogrammed death1 has a goodtherapeutic eï¬ectin lung cancer because carboplatin caninduce LINE1 expression Langer ThereforeLINE1 can be used as a target of combined immunotherapyin tumor therapyOther InhibitorsRecently a number of other regulatory approaches have beenreported In somatic cells microRNAs miRNAs or miRs alsoregulate the activity of LINE1 Idica MiR128regulates LINE1 activity in somatic cells by targeting the nuclearimport factor transportin1 TNPO1 3cid48UTR which mediatesnuclear import and requires RanGTP for cargo delivery into thenucleus Twyï¬els MiR128 inhibits the expression ofTNPO1 mRNA and protein and TNPO1 deficiency suppressesLINE1 mobilization by inhibiting nuclear import of LINE“RNP Idica MiR128 also guides the miRNAinduced silencing complex to bind directly to a target siteresiding in the ORF2 RNA of LINE1 Hamdorf At present a novel target of miR128 has been identified asheterogeneous nuclear ribonucleoprotein A1 hnRNPA1 whichis required for LINE1 retrotransposition Goodier Fung MiR128 represses hnRNPA1 mRNA andprotein by targeting the CDS of hnRNPA1 which interactswith LINE1 ORF1p via RNA bridge to promote LINE1mobilization Goodier This interaction results intranslational repression of the LINE1 retrotransposition therebyreducing the risk of LINE1mediated mutagenesis Pedersenand Zisoulis Therefore microRNAs can be a target forLINE1 inhibitionAryl hydrocarbon receptor AHR is a ligandactivatedtranscription factor that activates LINE1 expression Teneng AHR is overexpressed in breast and thyroid cancerssuggesting that these tumors also overexpress LINE1 Powell Lai found that biseugenol a novel AHRinhibitor impeded cancer growth and inhibited EMT in gastriccancer cells Lai These findings suggest that targetingAHR with small molecule inhibitors may be a novel therapeuticapproach ORF1P phosphorylation by protein kinase A is alsorequired for LINE1 Kinase inhibitors specifically designed totarget LINE1 ORF1P phosphorylation may be associated withinhibition of LINE1 Bojang Therefore there isroom for drug development research focusing on targeting andinhibiting LINE1 ORF1P phosphorylationCONCLUSIONThe activation of LINE1 retrotransposon is associated with avariety of human diseases and is involved in the occurrenceand progression of disease through retrotranspositiondependentand retrotranspositionindependent mechanisms Currently ithas even become a marker of tumorigenesis and prognosis andis related to immune regulation The eï¬ective inhibition ofLINE1 activation has become a treatment for some diseasesThe inhibition of LINE1 in animal experiments can inhibitFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cZhang et alLINE1 Retrotransposition in Human Diseasethe occurrence and development of tumors so the clinicalapplication of LINE1 inhibitors is imminent In addition toexploring some known inhibitors other mechanisms of LINE inhibition should also be explored We summarized therelationship between LINE1 and diseaserelated immunity andproposed that LINE1 may aï¬ect the immune status of thebody by regulating metabolismleading to poor prognosisMetabolic substances can aï¬ect the immune microenvironmentfor examplelactic acid can lead to immunosuppressivemicroenvironment leading to poor prognosis of tumors Thedysregulation of LINE1 can lead to the disorder of glucoseand lipid metabolism and the inhibition of glucose and lipidmetabolism may reverse the disease progression caused byLINE1 Now the antitumor eï¬ect of regulating the body™smetabolism has entered clinical trials such as the significanteï¬ect of metformin in the treatment of tumors Therefore themetabolic status of diseases caused by LINE1 can be checkedMetabolic therapy combined with LINE1 inhibitors may inhibitthe progression of LINE1 and may improve the immunemicroenvironment to achieve the optimal therapeutic eï¬ectAUTHOR CONTRIBUTIONSXZ wrote the RZ and JY reviewed and revised the All authors contributed to the and approved thesubmitted versionFUNDINGThis work was supported by the National Natural ScienceFoundation of China Grant Nos and National Science and Technology Support Program of ChinaGrant No 2018ZX09201015 and Project of Science andTechnology of Tianjin Grant No 18JCQNJC82700REFERENCESAllen E Horvath S Tong F Kraft P Spiteri E Riggs A D High concentrations of long interspersed nuclear element sequence distinguishmonoallelically expressed genes Proc Natl Acad Sci USA “ 101073pnas1737401100Aporntewan C Phokaew C Piriyapongsa J Ngamphiw C Ittiwut CTongsima S Hypomethylation of intragenic LINE1 repressestranscription in cancer cells through AGO2 PLoS One 6e17934 journalpone0017934Ardeljan D Taylor M S Ting D T and Burns K H The human longinterspersed element1 retrotransposon an emerging biomarker of neoplasiaClin Chem “ 101373clinchem2016257444Ardeljan D Wang X Oghbaie M Taylor M S Husband D DeshpandeV LINE1 ORF2p expression is nearly imperceptible in humancancers Mob DNA 101186s1310001901912Baba Y Watanabe M Murata A Shigaki H Miyake K Ishimoto T LINE1 hypomethylation DNA copy number alterations and CDK6amplification in esophageal squamous cell carcinoma Clin Cancer Res “ 10115810780432CCR131645Baba Y Yagi T Sawayama H Hiyoshi Y Ishimoto T Iwatsuki M Long interspersed element1 methylation level as a prognosticbiomarker in gastrointestinal cancers Digestion “ Babushok D V and Kazazian H H Jr Progress
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"P=0.055 active treatment”intervention 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. 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. 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 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 Melanie.Schellekensradboudumc.nl van den Hurk Desiree GM 2 Desiree.vandenHurkradboudumc.nl Prins Judith B 3 Judith.Prinsradboudumc.nl Molema Johan 2 Johan.Molemaradboudumc.nl Donders A Rogier T 4 Rogier.Dondersradboudumc.nl Woertman Willem H 4 Willem.Woertmanradboudumc.nl van der Drift Miep A 2 Miep.vanderDriftradboudumc.nl Speckens Anne EM 1 Anne.Speckensradboudumc.nl 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.; licensee BioMed Central Ltd. This is an open access distributed under the terms of the Creative Commons Attribution License (http://creativecommons./licenses/by/2.0) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. Background Lung cancer is the leading cause of cancer death worldwide and characterized by a poor prognosis. It has a major impact on the psychological wellbeing of patients and their partners. Recently it has been shown that Mindfulness-Based Stress Reduction (MBSR) is effective in reducing anxiety and depressive symptoms in cancer patients. The generalization of these results is limited since most participants were female patients with breast cancer. Moreover only one study examined the effectiveness of MBSR in partners of cancer patients. Therefore in the present trial we study the effectiveness of MBSR versus treatment as usual (TAU) in patients with lung cancer and their partners. Methods/Design A parallel group randomized controlled trial is conducted to compare MBSR with TAU. Lung cancer patients who have received or are still under treatment and their partners are recruited. Assessments will take place at baseline post intervention and at three-month follow-up. The primary outcome is psychological distress (i.e. anxiety and depressive symptoms). Secondary outcomes are quality of life (only for patients) caregiver appraisal (only for partners) relationship quality and spirituality. In addition cost-effectiveness ratio (only in patients) and several process variables are assessed. Discussion This trial will provide information about the clinical and cost-effectiveness of MBSR compared to TAU in patients with lung cancer and their partners. Trial registration ClinicalTrials.gov NCT01494883. Mindfulness-based stress reduction Lung cancer patients Partners Psychological distress Randomized controlled trial Background With an estimated 1.4 million deaths per year lung cancer is the leading cause of death by cancer worldwide. Even with the best available treatment five-year survival is merely 16% and about 60 to 70% of patients die within the first year after diagnosis [1]. This poor prognosis is often caused by a late diagnosis as the presentation usually occurs when the lung cancer is advanced. Patients may develop burdensome symptoms like pain dyspnoea fatigue and cough and they may undergo radical treatment including surgery chemo- and radiotherapy. Not surprisingly lung cancer has a major impact on the psychological wellbeing of patients and their family. Akechi and colleagues [2] showed that 19% of patients with advanced lung cancer meets the criteria of psychiatric disorders especially depressive and adjustment disorders. Of patients who had been successfully treated for lung cancer 15% met the criteria for a minor or major depressive disorder [3]. The prevalence rate of depressive and anxiety symptoms among lung cancer patients ranges from 20 to 47% [4-7]. Compared to patients with other cancer diagnoses lung cancer patients report the highest rates of distress (43 to 58%) [89] resulting in a lower quality of life [10]. Family friends and especially partners of patients with lung cancer also have to deal with its psychological impact [11-14]. Partners not only provide emotional and practical support they also have to cope with their own concerns including the uncertainty regarding the course of the illness and the fear of losing their partner [15]. More than 50% of partners of lung cancer patients report negative emotional effects of caregiving [16]. Around 40% of partners of patients with advanced lung cancer report high levels of distress [17]. The relationship between patient and partner can also be affected by the cancer. It has been shown that some partners report a lower quality of their relationship after the diagnosis of lung cancer [18]. Though numerous studies examined the psychological distress of lung cancer patients and their partners [2-22] not much research is done on how to alleviate distress in these groups [23]. In addition the available studies on managing the psychosocial care needs of cancer patients and their families have focused on care at the very end of life (e.g. [24-26]). Recently studies have demonstrated that palliative care initiated early in treatment improves the quality of life and depressive symptoms of lung cancer patients [1027]. This stresses the importance of integrating psychosocial care for lung cancer patients and their partners early in the treatment rather than instigating it once life-prolonging therapies fail. In the past ten years MBSR has become a promising psychosocial intervention for cancer patients. Mindfulness is defined as intentionally paying attention to moment-by-moment experiences in a non-judgmental way [28]."
1
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 People™s 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 manufacturer™s 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] Children™s 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 patient™s 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 manufacturer™s 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 patient™s 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 × 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 Agilent™s 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 × 109L with blasts a hemoglobin count of a0 gL and a platelet count of × 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 French“American“Britishh 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 People™s 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 People™s Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr “Luppi M 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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 “ DíazBeyá 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 Styczyński 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 “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'
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"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 people™s republic of china2institute of clinical pharmacology engineering research center for appliedtechnology of pharmacogenomics of ministry of education central southuniversity changsha people™s 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 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 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 chen™s 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 œheadtohead alignment nextto the dna and thus form stable œladderlike 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 sarcoma“associated 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 cgas“sting 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 cell“like 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 in œopenconformation submicromolarlevels induce sting activationand ifn productionhuman fibroblastsantiviral activity colon tumors mgkg iv of a treatedgroup remainedtumor free faa flavone acetic acid dmxaa 56dimethylxanthenone4acetic acid cma 10carboxymethyl9acrid
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"Surgical resections can be indicated for localized mediastinal or lung lesions. Other treatments include low-fat medium-chain triglyceride diets interferon-alpha radiation corticosteroids chemotherapy somatostatin and propranolol (2). We revealed an unusual presentation of DPL in a middle-aged asymptomatic woman. In follow-up imaging studies the extent of the disease had increased and a surgical biopsy was performed to rule out lymphangitic carcinomatosis. An awareness of the possibility of DPL even in adults is necessary. 1 Swensen SJ Hartman TE Mayo JR Colby TV Tazelaar HD M¼ller NL Diffuse pulmonary lymphangiomatosis: CT findings J Comput Assist Tomogr 1995 19 348 352 7790540 2 Yoo SH Song JS Lee JJ Lee M Hwang HS Jang SJ Diffuse pulmonary lymphangiomatosis: Pulmonary lymphatic disorder in an adult Basic Appl Pathol 2012 5 63 66 3 El Hajj L Mazi¨res J Rouquette I Mittaine M Bolduc JP Didier A Diagnostic value of bronchoscopy CT and transbronchial biopsies in diffuse pulmonary lymphangiomatosis: case report and review of the literature Clin Radiol 2005 60 921 925 16039928 4 Boland JM Tazelaar HD Colby TV Leslie KO Hartman TE Yi ES Diffuse pulmonary lymphatic disease presenting as interstitial lung disease in adulthood: report of 3 cases Am J Surg Pathol 2012 36 1548 1554 22982897 5 Du MH Ye RJ Sun KK Li JF Shen DH Wang J Diffuse pulmonary lymphangiomatosis: a case report with literature review Chin Med J (Engl) 2011 124 797 800 21518582 6 Resten A Maitre S Humbert M Rabiller A Sitbon O Capron F Pulmonary hypertension: CT of the chest in pulmonary venoocclusive disease AJR Am J Roentgenol 2004 183 65 70 15208112 7 Wittenberg KH Swensen SJ Myers JL Pulmonary involvement with Erdheim-Chester disease: radiographic and CT findings AJR Am J Roentgenol 2000 174 1327 1331 10789787 8 Copley SJ Coren M Nicholson AG Rubens MB Bush A Hansell DM Diagnostic accuracy of thin-section CT and chest radiography of pediatric interstitial lung disease AJR Am J Roentgenol 2000 174 549 554 10658741 9 Nobre LF M¼ller NL de Souza Jºnior AS Marchiori E Souza IV Congenital pulmonary lymphangiectasia: CT and pathologic findings J Thorac Imaging 2004 19 56 59 14712135 10 Tazelaar HD Kerr D Yousem SA Saldana MJ Langston C Colby TV Diffuse pulmonary lymphangiomatosis Hum Pathol 1993 24 1313 1322 8276379 Fig. 1 52-year-old woman with diffuse pulmonary lymphangiomatosis. Posteroanterior chest radiograph revealing increased interstitial markings in both lungs (A). Initial CT scan demonstrating diffuse interstitial thickening in both lungs (B C) and lymph node enlargement in right anterior diaphragmatic area (D arrow). Follow-up CT taken eight months after initial CT scan showing increased interlobular septal thickening (E F) with more prominent bronchovascular bundle thickening (arrows) and increased bilateral pleural effusion (G). Intra-operative photograph showing hypervascularity of lung surface (H). I. Hematoxylin and eosin-stained section of lesion showing proliferation of thin-walled anastomosing lymphatic vessels lined by single layer of endothelial cells lacking cytological atypia (arrows — 200). J. Immunohistochemical staining with D2-40 revealing proliferative lymphatic channels (arrows — 200). Summary of Previously Reported Middle-Aged Patients with Pulmonary Lymphangiomatosis 2984705R 2786 Cancer Res Cancer Res. Cancer research 0008-5472 1538-7445 24648348 4278592 10.1158/0008-5472.CAN-13-3157 EMS57520 EGFR-Mediated Chromatin Condensation Protects KRAS-Mutant Cancer Cells Against Ionizing Radiation Wang Meng 1 Kern Ashley M. 1 H¼lsk¶tter Marieke 1 Greninger Patricia 2 Singh Anurag 2 Pan Yunfeng 3 Chowdhury Dipanjan 3 Krause Mechthild 4 5 6 7 Baumann Michael 4 5 6 7 Benes Cyril H. 2 Efstathiou Jason A. 1 Settleman Jeff 2 * Willers Henning 1 * 1Department of Radiation Oncology Massachusetts General Hospital Harvard Medical School 55 Fruit Street Boston Massachusetts 02114 2Center for Cancer Research Massachusetts General Hospital Cancer Center Harvard Medical School 149 13th Street Charlestown Massachusetts 02129 3Department of Radiation Oncology Dana-Farber Cancer Institute Harvard Medical School 450 Brookline Ave Boston Massachusetts 02115 4Department of Radiation Oncology Medical Faculty and University Hospital Carl Gustav Carus Technische Universitt Dresden Dresden Germany 5OncoRay- National Center for Radiation Research in Oncology Medical Faculty and University Hospital Carl Gustav Carus Technische Universitt Dresden Helmholtz-Zentrum Dresden-Rossendorf Dresden Germany 6Institute of Radiation Oncology Helmholtz-Zentrum Dresden-Rossendorf Dresden Germany 7Cancer Consortium (DKTK) partner site Dresden and German Cancer Research Center (DKFZ) Heidelberg Germany Corresponding Author: Henning Willers M.D. Dept. of Radiation Oncology Massachusetts General Hospital 55 Fruit Street Boston MA 02114. Tel. 617-726-5184 hwillerspartners. * co-senior authors 31 3 2014 19 3 2014 15 5 2014 30 12 2014 74 10 2825 2834 Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non-small cell lung cancer cell (NSCLC) lines we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in-vitro and in-vivo thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like co-localization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is co-dependent upon EGFR and PKC?. PKC? in addition to MEK/ERK signaling is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. "
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